|MO NAMAN ATTITUUNIKAUS009962450B2 MITATTI (12 ) United States Patent (10 ) Patent No. : US 9 , 962, 450 B2 Kraynov et al. (45 ) Date of Patent : May 8 , 2018

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References Cited Wells , JA et al. , “ Importance of hydrogen -bond formation in sta ( 56 ) bilizing the transition state of subtilisin , ” Phil Trans R Soc Lond A OTHER PUBLICATIONS 1986 ; 317 :415 -423 . Woghiren , C et al. , “ Protected thiol- polythylene glycol: a new Van Hest, JC et a ., “ Efficient Incorporation of Unsaturated Methio activated polymer for reversible protein modification , " Bioconjug Chem . Sep . - Oct. 1993 ; 4 ( 5 ) :314 - 8 . nine Analogues into Proteins in Vivo ," J Am Chem Soc Wong , SS and LJ Wong , “ Chemical crosslinking and the stabiliza 2000 ;122 (7 ): 1282 - 1288 . tion of proteins and enzymes ,” Enxyme Microb Technol. Nov. 1992 ; Van Solingen , P and JB van der Plaal, “ Fusion of yeast 14 ( 11 ) : 866 - 74 . spheroplasts ,” J Bacteriol . May 1977; 130 ( 2 ): 946 - 7 . Wright, K , “ Biotechnology : Insect virus as super- vector ? ,” Nature Veronese , FM et al ., “ Surface modification ofproteins . Activation of ( 1986 ) 321 (6072 ) :718 . monomethoxy -polyethylene glycols by phenylchloroformates and Wu, et al. , “ Enzymatic Phosphorylation of Unnatural Nucleosides ” , modification of ribonuclease and superoxide dismutase ,” Appl J . Am . Chem . Soc . ( 2002 ) 124 ( 49 ) : 14626 - 14630 . Biochem Biotechnol. Apr. 1985 ; 11 ( 2 ) : 141 -52 . Yelton , MM et al ., “ Transformation of Aspergillus nidulans by using Vlak , JM et al. , “ Functional studies on the p10 gene of Autographa a trpC plasmid ,” Proc Natl Acad Sci USA .Mar . 1984 ;81 (5 ): 1470 -4 . californica nuclear polyhedrosis virus using a recombinant express Yelverton , E et al. , “ Bacterial synthesis of a novel human leukocyte interferon , ” Nucleic Acids Res. Feb . 11 , 1981; 9 ( 3 ) :731 -41 . ing a p10 -beta - galactosidase fusion gene , ” J Gen Virol. Apr. Zalipsky , S et al, “ Attachment of drugs to polyethylene glycols ,” 1988 ;69 ( P14 ): 765 -76 . Eur Polymer Journal. 1983 ;19 ( 12 ): 1177 - 83 . Wang , L and PG Schulz , “ Expanding the genetic code ,” Chem Zalipsky , S et al. , “ Functionalized Poly ( ethylene glycol ) for prepa Commun (Camb ). Jan . 7, 2002 ;1 : 1 - 11. ration of biologically relevant conjugates , " Bioconjug Chem . Mar . Wang, L et al. , “ Addition of the keto funcitonal group to the genetic Apr . 1995 ; 6 ( 2 ) : 150 -65 . code of Escherichia coli ,” Proc Natl Acad Sci. ( 2003 ); 100 ( 1 ): 56 -61 . Zalipsky, S ., et al. , “ Use of Functionalized Poly (Ethylene Glycols ) Wang , L et al ., “ Expanding the genetic code of Escherichia coli, ” for Modification of Polypeptides ” in Polyethylene Glycol Chemis Science. Apr. 20 , 2001 ; 292 (5516 ): 498 -500 . try : Biotechnical and Biomedical Applications , Hanis , Plenus Press , Wang , W , “ Instability , stabilization , and formulation of liquid pro New York ( 1992 ), and in Advancing Drug Reviews, 16 : 157 -182 tein pharmaceuticals, ” Intl Pharm . Aug. 20 , 1999 ; 185 ( 2 ): 129 -88 . ( 1995 ) . Wang , Q , et al. , “ Bioconjugation by Copper ( I ) -Catalyzed Azide Zhang , Z et al. , “ A new strategy for the site- specific modificaiton of Aikyne [ 3 + 2 ] Cycloaddition , ” J Am Chem Soc 2003 ; 125 ( 11) : 3192 proteins in vivo ,” Biochemistry. Jun . 10 , 2003 ;42 (22 ): 6735 - 46 . 3193 . Zoller , MJ and M Smith , “ Oligonucleotide - directed mutagenesis : a Weiss , " Relaxin ” , Ann . Rev . Physiol. , ( 1984 ) 46 :43 - 52 . simple method using two oligonucleotide primers and a single Weissman , C , “ The cloning of interferon and other mistakes , ” in stranded DNA template, ” Methods Enzymol . 1987; 154 : 329 -50 . Interferon 3 1981; ed . I. Gresser ; Academic Press , London , 101- 134 . Zoller, MJ and M Smith , “ Oligonucleotide -directed mutagenesis Weitkamp et al. , “ Additional Data on the Population Distribution of using M13 - derived vectors: an efficient and general procedure for Human Serum Albumin Genes ; Three New Variants ” , Ann . Hum . the produciton of point mutations in any fragment of DNA ” Nucleic Genet. ( 1973 ) 37 :219 - 26 . Acids Res. Oct . 25 , 1982 ; 10 ( 20 ) :6487 -500 . Wells , JA et al. , “ Cassette mutagenesis : an efficient method for Zoller , MJ & M . Smith , “ Oligonucleotide - directed mutagenesis of generation of multiple mutations at defined sites, ” Gene . 1985 ;34 ( 2 DNA fragments cloned into M13 vectors ” , Methods Enzymol. 3 ) :315 -23 . 1983 ; 100 : 468 -500 . U . S . Patent May 8 , 2018 Sheet 1 of 26 US 9 , 962, 450 B2

Figure 1

Light gray = chain A Street Dark gray = chain B Black = selected sites

V - BA17

.

.

AV13 . AV130 . B W27 . : BV6 6RLX BE4 Insulin numbering BS1 Site not in structure : A Q - 3 U . S . Patent May 8 , 2018 Sheet 2 of 26 US 9 , 962, 450 B2

Figure 2

Light gray = chain A Dark gray = chain B Black = selected sites

O AA5

*

*

* AV13 BW27BW27A 5 A R18 TO BV6 ORLX BE4 Insulin numbering BS1 Site not in structure : A Q - 3 U . S . Patent May 8, 2018 Sheet 3 of 26 US 9 , 962, 450 B2

Figure 3

Lightblue = chain A Dark blue = chain B Yellow = receptor binding site Orange = selected sites • BA17

AV131 BV6 AAS A AL2 BE4E BS1 A R18 BW27 6RLX Insulin numbering Site not in structure : A Q - 3 U . S . Patent May 8 , 2018 Sheet 4 of 26 US 9 , 962, 450 B2

Figure 4

NHg+

COO 00000000 90000000 B29

800000000 B15 000000000000B20 U . S . Patent May 8, 2018 Sheet 5 of 26 US 9 , 962, 450 B2

Figure 5

1 2 3 4 5 Lane Sample sir . SeeBlue2 MW Marker RLX - EX -002 - 129 : Oh nariadhadisabilitijela RLX - EX - 002- 129 : 2h wa RLX - EX -002 - 129 : 4h UAWNH RLX -EX -002 - 129 : 6h

1

: 1 SHADEMA Pro -Relaxin - BA1 -AV13pAF U . S . Patent May 8, 2018 Sheet 6 of 26 US 9 , 962, 450 B2

Figure 6A SDS -PAGE ? unPEGylated Relaxin Da 195

| Lane Lane assignment

o MW Marker

N titN + wt Relaxin (NR ) - - - 33 A - Chain& B - Chain con Lane # : 1 2 3 4 5 6 7 6 7 wt Relaxin ( R )

Figure 6B SDS -PAGE ? 20K PEGylated Relaxin 1011 % 193

93 Lane Lane assignment

1 MW Marker

20K PEG Relaxin thedown (NR ) - B -Chain 20K PEG Relaxin (R ) Lane # : 1 2 3 4 U . S . Patent May 8 , 2018 Sheet 7 of 26 US 9 , 962, 450 B2

Figure 7

PK evaluation of PEG size 10007 PEG3OK -AV13 - PEG2OK - AV3 + PEG1OK - AV13 SerumRelaxinConc.Ingim) + PEGSK -AV13 WT Relaxin

2006040 60 0700100 Time ( hrs ) U . S . Patent May 8 , 2018 Sheet 8 of 26 US 9 , 962, 450 B2

Figure 8A Mean RLX serum concentration versus time Error bars = Std Dev , Gender = males Dose = 0 .5mg /kg , Route - SC 1000, + RLX - A - AQ1- 20K SerumRelaxinConc.(ng/mL) + RLX - A - AA5 - 20K 1 +T1T RLX - A - AR18 - 20K A140 RLX - A -BV7 - 20K - RLX - A -BW28 - 20K FH-. o RLX - A -AV13 - 20K

20 40 60 80 100 Time (hrs ) Figure 8B Individual PEG20 - AQ1- RLX serum concentration versus time Gender = males Dose = 0 .5mg / kg , Route - SC 10007 An1 . An2 SerumRelaxinConc.(ng/mL) An3 And . An5

To 20 40 60 80 100 Time (hrs ) U . S . Patent May 8 , 2018 Sheet 9 of 26 US 9, 962 , 450 B2

Figure 9A

Individual PEG20 - AA5 -RLX serum concentration versus time Gender= males Dose = 0 .5mg / kg , Route - SC 10007

Ant

SerumRelaxinConc.(ng/mL) E. An2 A An3 V An4

I Anasse) An5

0 20 40 60 80 100 Time (hrs ) Figure 9B Individual PEG20 - AR18 -RLX serum concentration versus time Gender = males Dose = 0 . 5mg/ kg , Route - SC 10007

SerumRelaxinConc.(ng/mL) 100 Ant 1 An2 An3 " AndAn4 AngAn5

1 0+ 20 40 60 80 100 Time (hrs ) U . S . Patent May 8 , 2018 Sheet 10 of 26 US 9 , 962 ,450 B2

Figure 10A Individual PEG20- BV7 -RLX serum concentration versus time Gender = males Dose = 0 .5mg / kg , Route - SC 1000

SerumRelaxinConc.(ng/ml) 8 An1 An2 It An3 ; An4 An5 L 0 20 40 60 80 100 Time (hrs ) Figure 10B Individual PEG20 - BW28 -RLX serum concentration versus time Gender =males Dose = 0 . 5mg/ kg , Route = SC 10007

SerumRelaxinConc.(ng/mL) 100 - An1 1 An2 A An3 y An4 An5

1+ 0 20 40 60 80 100 Time (hrs ) U . S . Patent May 8 , 2018 Sheet 11 of 26 US 9 , 962 ,450 B2

Figure 11

Individual PEG20 - AV13 -RLX serum concentration versus time Gender = males Dose = 0 . 5mg /kg , Route - SC 10000

000 SerumRelaxinConc.(ng/mL) An1 An2 in An3 An4 An5

o 20 40 60 80 100 Time (hrs ) U . S . Patent May 8, 2018 Sheet 12 of 26 US 9 ,962 ,450 B2

Figure 12A Mean RLX serum concentration versus time Error bars = Std Dev , Gender= males Dose = 0 .5mg / kg , Route - SC 1000

+ WT Relaxin SerumRelaxinConc.(ng/mL) ok

Time (hr ) Figure 12B Individual wt rhRelaxin serum concentration versus time Gender = males Dose = 0 .5mg /kg , Route = SC 1000 An1 SerumRelaxinConc.(ng/mL) An2 A An3 An4 An5

etõ Time (hr ) U . S . Patent May 8, 2018 Sheet 13 of 26 US 9 ,962 ,450 B2

Figure 13A

Mean RLX serum concentration versus time Error bars = Std Dev , Gender= males Route = IV , SC 10000 1000 + IV - 0 . 25 MPK SerumRelaxinConc.(ng/mL) + SC - 0 .5 MPK 100 + SC - 0 .25 MPK + SC - 0 . 125 MPK

Ì

Àto 20 40 60 80 100 Time (hr ) Figure 13B Individual RLX serum concentration versus time Gender - males Route = IV , Dose = 0 . 25 MPK 10000

• An1 I An2 SerumRelaxinConc.(ng/mL) An3 An4 Hii!

o 40 60 80 100 Time (hr ) Too U . S . Patent May 8, 2018 Sheet 14 of 26 US 9 , 962 ,450 B2

Figure 14A

Individual RLX serum concentration versus time Gender = males Route = SC , Dose = 0 .5 MPK 000 An1 An2 SerumRelaxinConc.(ng/mL) 100 An3 And An5

o 20 40 60 80 100 Time (hr ) Figure 14B Individual RLX serum concentration versus time Gender = males Route = SC , Dose = 0 .25 MPK 1000 An1 An2 SerumRelaxinConc.(ng/mL) An3

20 40 60 80 100 Time (hr ) U . S . Patent May 8, 2018 Sheet 15 of 26 US 9 , 962 ,450 B2

Figure 15

Individual RLX serum concentration versus time Gender = males Route = SC , Dose = 0 . 125 MPK 1007 Ant | An2 SerumRelaxinConc.(ng/mL) An3 An4 : An5

0 20 40 60 80 100 Time (hr ) U . S . Patent May 8, 2018 Sheet 16 of 26 US 9 , 962 ,450 B2

Figure 16A

Mean Terminal Half -life values versus dose Error bars = Std Dev Route = SC , Gender = males

? TerminalHalf-life(hr) 0

0 . 2 0 . 4 0 . 6 Dose (mg / kg ) Figure 16B Mean AUCinf versus dose Error bars = Std Dev , Route = SC Species = SD rats , Gender = males 15000

RelaxinAUCinf(ng*hr/mL) 10000 I

0 .2 0. 4 0 .6 Dose (mg / kg ) U . S . Patent May 8, 2018 Sheet 17 of 26 US 9 , 962 ,450 B2

Figure 17A

Mean Cmax versus dose Error bars = Std Dev , Route = SC Species = SD rats , Gender= males

SerumRelaxinCmax(ng/mL)

1

: 0 . 2 0 . 4 060. 6 Dose (mg / kg ) Figure 17B Mean Clearance versus dose Error bars = Std Dev , Route = SC Species = SD rats , Gender = males

RelaxinClearance(mL/hrkg)

00 !0 . 2! 00 . 4 !0 . 6 Dose (mg / kg ) U . S . Patent May 8, 2018 Sheet 18 of 26 US 9 , 962 ,450 B2

Figure 18A

Mean Volume of Distribution values versus dose Error bars = Std Dev Route = $ C , Gender - males 900

800 RelaxinVolumeofDistribution (mL/kg)

400 + 0 . 0 0 . 2 0. 4 0 . 6 Dose (mg / kg ) Figure 18B Mean RLX serum concentration versus time Error bars = Std Dev , Gender = males Open symbols = IV , Closed symbols = SC 100007

SerumRelaxinConc.(ng/mL)

20 40 60 80 100 Time (hr ) U . S . Patent May 8 , 2018 Sheet 19 of 26 US 9 , 962 ,450 B2

Figure 19A

Effect ofWild - Type Relaxin IV Infusion on Water Intake and Urine Output in Female Long - Evans Rats

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Water Intake ( 0 - 6 br) Baseline Urine ( - 16 -Ohr ) Urine ( 0 - 6 hr )

Figure 19B

Baseline Urine ( - 16 - 0 hr ) in Female Long - Evans Rats Volume(mL/100gBW) MWAND Vehicle 0 . 3 X U . S . Patent May 8, 2018 Sheet 20 of 26 US 9 , 962 ,450 B2

Figure 19C

Effect of Wild -Type Relaxin IV Infusion on Hematocrit in Female Long -Evans Rats .. . . mean + / - sem n = 4 . : : : : : ......

Hematocrit(%) ñ n Vehicle 10. 3X ? 01X

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Figure 19D

Effect of Wild - Type Relaxin IV Infusion On Plasma BUN In Female Long - Evans Rats

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Ohr 2 hr 6 hr U . S . Patent May 8, 2018 Sheet 21 of 26 US 9 , 962 ,450 B2

Figure 19E

Effect of Wild - Type Relaxin IV Infusion on Water Intake ( 0 - 6 hr) in Female Long - Evans Rats

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Figure 19F

Effect of Wild - Type Relaxin IV Infusion on Urine Output (0 -6hr ) in Female Long -Evans Rats

Volume(mL/100gBW) - - f. Vehicle 0. 3 x 1x 10 x U . S . Patent May 8, 2018 Sheet 22 of 26 US 9 , 962 ,450 B2

Figure 20A

Effect of PEG - Relaxin Variant IV Administration On Water Intake and Urine Output In Female Long - Evans Rats 6 . 00 . . . . .* * * mean + / - sem , n = 4 5 . 00 ...... n Vehicle 4 .00 ...... det Volume(mL/100gBW) * 0 . 1X 3 .00 * 0 .3 X 2 .00 ...... S . SALO ...... X

1 .00 . . . 0 . 00 + Water Intake ( 0 - 6hr) Baseline Urine( - 16 - Ohr ) Urine (0 - 6hr )

Figure 20B

Effect of PEG - Relaxin Variant IV Administration On Plasma Sodium In Female Long - Evans Rats

142 ...... WAY ! ! . . " VM ' . . ." ! " . . " ' . . mean + / - sem , n = 3 - 4 140 wwwWit ' . seved Vehicle ASTERS WISATA Sodium(mEq/L) 138 SwedbankW . WAN " ...... 0 . 1X 136 Twife 0 .3 pw Paziticha 7 . GEWSWWWpentadbiran CHAMA WW 134 www 1X wwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwwww www 132 130 + Ohr 2 hr 6 hr U . S . Patent May 8, 2018 Sheet 23 of 26 US 9 , 962 ,450 B2

Figure 20C Effect of PEG -Relaxin Variant IV Administration On Plasma Nat Change In Female Long -Evans Rats mean I sem , n = 3 - 4 No# - Vehicle w 0 . 1 . . VerOhr ...... trohimona : s 0. 3X WAY www...... PlasmaNatChangefromBaseline erio energie 1X i ...... - ...... Whribariyle ......

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Figure 20E

WANAMW het Effect of PEG -Relaxin Variant IV Administraton on Plasma Osmolarity Change in Female Long - Evans Rats 10 mean I sem , n = 3 - 4 ...... NEWS

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Effect of PEG - Relaxin Variant IV Administration on BUN in Female Long - Evans Rats 30 . . mean I sem , n = 4

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Ohr 2 hrhr 6 hr U . S . Patent May 8, 2018 Sheet 25 of 26 US 9 , 962, 450 B2

Figure 20G Effect of PEG -Relaxin Variant IV Administration on Water Intake (0 -6hr ) in Female Long - Evans Rats

WaterIntake(mL/100gBW)prooniai toof

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Volume(ml/100gBV ? • oo

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Vehicle 0 . 1X 0 . 3X U . S . Patent May 8, 2018 Sheet 26 of 26 US 9 , 962 ,450 B2

Figure 201

Effect of PEG -Relaxin Variant IV Administration on Urine Output (0 -6hr ) in Female Long -Evans Rats

Volume(mL/100gBW)

Vehicle 0. 1 % 0. 3x x US 9 , 962 , 450 B2

METHOD OF TREATING HEART FAILURE changes, together with the increased sympathetic stimula WITH MODIFIED RELAXIN POLYPEPTIDES tion of the heart , lead to further and often decompensating demands on the remaining functional myocardium . CROSS REFERENCE TO RELATED Congestive heart failure, which is a common end point for APPLICATIONS 5 many cardiovascular disorders , results when the heart is unable to adequately perfuse the peripheral tissues . Accord This application is a divisional of U . S . patent application ing to recent estimates , there are about 4 million people in Ser . No. 14 / 152 ,302 , filed Jan . 10 , 2014 , which is a divi the United States diagnosed with this disease , and more than sional of U . S . patent application Ser. No . 13 /211 , 101 , filed 50 % of these cases are fatal within 5 years of diagnosis Aug . 17 , 2011 . now U . S . Pat . No . 8 ,735 , 539 , which claims 10 [ Taylor, M . D . et al. , Annual Reports in Med . Chem . 22 , the benefit of U . S . Provisional Patent Application No. 85 - 94 ( 1987 ) ). 61/ 374 ,582 , filed Aug . 17 , 2010 , each of which is hereby Current therapy for heart failure , including congestive incorporated by reference in its entirety . heart failure , focuses on increasing cardiac output without causing undue demands on the myocardium . To achieve SEQUENCE LISTING 15 these ends , various combinations of diuretics , vasodilators and inotropic agents are used to decrease blood volume, to This application includes a sequence listing which has decrease peripheral resistance , and to increase force of been submitted via EFS -Web in a file named cardiac contraction . Current therapy therefore depends on “ 4327001205 . txt” created on Aug . 17 , 2016 and having a balancing the effects of multiple drugs to achieve the clinical size of 50 , 246 bytes , which is hereby incorporated by 20 needs of individual patients , and is plagued by adverse reference in its entirety . reactions to the drugs used . For example , diuretics decrease plasma concentrations of FIELD OF THE INVENTION potassium and magnesium and increase the incidence of arrhythmias in patients receiving digitalis . Diuretics can This invention relates to relaxin polypeptides optionally 25 potentiate the circulatory effects of nitrates through volume modified with at least one non -naturally encoded amino depletion and lead to decreases in filling pressure of the acid . heart , cardiac output and systemic arterial pressure . Alpha adrenergic antagonists can lead to marked falls in BACKGROUND OF THE INVENTION systemic arterial pressure that compromise coronary perfu 30 sion . Mature human relaxin is a hormonal peptide of approxi- Angiotensin converting enzyme inhibitors can have simi mately 6000 daltons known to be responsible for remodel- lar effects on arterial pressure and additionally lead to ling the reproductive tract before parturition , thus facilitat- excessive increases in plasma concentrations of potassium . ing the birth process . This protein appears to modulate the Drugs that lead to positive inotropy, such as digitalis and restructuring of connective tissues in target organs to obtain 35 beta adrenergic antagonists , have the potential to provoke the required changes in organ structure during pregnancy arrhythmias . In addition , digitalis has a narrow therapeutic and parturition . See , Hisaw , F . L ., Proc . Soc . Exp . Biol. index and the catecholamine analogs all tend to loose their Med ., 23 : 661 - 663 (1926 ); Schwabe, C . , et al ., Biochem . effectiveness rapidly , due to receptor downregulation . Biophys . Res. Comm . , 75 : 503 - 570 ( 1977 ) ; James, R . et al. , Thus there is a need for therapeutic agents that lead to Nature , 267 : 544 - 546 ( 1977 ) . A concise review of relaxin 40 physiologically integrated responses of arterial and venous was provided by Sherwood , D . in The Physiology of Repro vasodilation and cardiac inotropy, and are devoid of the duction , Chapter 16 , “ Relaxin ” , Knobil , E . and Neill , J ., et disadvantages of the currently used therapeutic agents . al. ( eds. ), (Raven Press Ltd ., New York ) , pp . 585 -673 Relaxin has been purified from a variety of species ( 1988 ) . Circulating levels of relaxin are elevated for the including porcine , murine, equine, shark , tiger, rat, dogfish entire ninemonths of pregnancy and drop quickly following 45 and human , and shows at least primary and secondary delivery . structural homology to insulin and the insulin - like growth While predominantly a hormone of pregnancy , relaxin has factor, however homology between species can be quite low . also been detected in the non -pregnant female as well as in In the human , relaxin is found in most abundance in the the male . Bryant- Greenwood , G . D . , Endocrine Reviews, 3 : corpora lutea ( CL ) of pregnancy . However, specific nuclei in 62 - 90 (1982 ) and Weiss , G ., Ann . Rev. Physiol. , 46 : 43 -52 50 the brain have relaxin receptors and other nuclei contain ( 1984 ) and has most recently been found to be useful in the messenger RNA for relaxin . Several nuclei with cells bear treatment of heart failure . ing relaxin receptors are found in the area of the hypothala Heart failure is defined as the inability of the cardiac mus. pump to move blood as needed to provide for the metabolic Two human gene forms have been identified , (H1 ) and needs of body tissue . Decreases in pumping ability arise 55 (H2 ) . Hudson , P ., et al ., Nature , 301 : 628 -631 ( 1983 ) ; mostm often from loss or damage ofmyocardial tissue . As a Hudson , P ., et al. , The EMBO Journal , 3 : 2333 - 2339 (1984 ) ; result , ventricular emptying is suppressed which leads to an and U . S . Pat. Nos . 4 , 758 , 516 and 4 , 871 ,670 . Only one of the increase in ventricular filling pressure and ventricular wall gene forms (H2 ) has been found to be transcribed in CL . It stress , and to a decrease in cardiac output. As a physiological remains unclear whether the (H1 ) form is expressed at response to the decrease in cardiac output, numerous neu - 60 another tissue site , or whether it represents a pseudo - gene. roendocrine reflexes are activated which cause systemic When synthetic human relaxin (H2 ) and certain human vasoconstriction , sympathetic stimulation of the heart and relaxin analogs were tested for biological activity , the tests fluid retention . Although these reflex responses tend to revealed a relaxin core necessary for biological activity as enhance cardiac output initially , they are detrimental in the well as certain amino acid substitutions for methionine that long term . The resulting increases in peripheral resistance 65 did not affect biologicalactivity . Johnston , et al. , in Peptides: increase the afterload on the heart and the increases in blood Structure and Function , Proc . Ninth American Peptide Sym volume further increase ventricular filling pressure . These posium , Deber , C . M ., et al. ( eds. ) (Pierce Chem . Co . 1985 ) . US 9 , 962, 450 B2 Methods ofmaking relaxin are also described in U . S . Pat . coronary artery disease , scleroderma, stroke, diastolic dys No . 4 ,835 , 251 and in co - pending U . S . Ser . No. 07 / 908 , 766 function , familial hypercholesterolemia , isolated systolic ( PCT US90 /02085 ) and Ser. No . 08 / 080 , 354 (PCT US94 / hypertension , primary hypertension , secondary hyperten 0699 ). Methods of using relaxin in cardiovascular therapy sion , left ventricular hypertrophy, arterial stiffness associ and in the treatment of neurodegenerative diseases are 5 ated with long - term tobacco smoking , arterial stiffness asso described in U .S . Pat . No . 5 , 166 , 191 and in U . S . Ser. No . ciated with obesity , arterial stiffness associated with age , 07 / 902 ,637 (PCT US92 / 06927 ) . Certain formulations of systemic lupus erythematosus , preeclampsia , and hypercho human relaxin are described in allowed U . S . Ser. No. lesterolemia . In related embodiments , the invention provides 08 /050 ,745 . methods of increasing arterial compliance in perimeno Recombinant human relaxin (H2 ) in currently in Phase I 10 pausal, menopausal, and post -menopausal women and in human clinical trials in scleroderma patients. Scleroderma is individuals who are at risk of one of the aforementioned a disease involving an imbalance in tissue reformation disorders . giving rise to the overproduction of collagen , and ultimately In an additional embodiment of the invention , adminis resulting in swelling and hardening of the skin ( and affected tration of relaxin increases arterial compliance by at least organs ). Currently treatments delivering relaxin require 15 10 % , 15 % , 20 % or more , relative to the measured arterial repeated and prolonged infusions. compliance before administration . In still further embodi ments , the invention provide for the administration of SUMMARY OF THE INVENTION relaxin to individuals with diminished arterial compliance at a predetermined rate so as to maintain a serum concentration The invention provides relaxin polypeptides optionally 20 of relaxin from 0 . 5 to 80 ng /ml . In one embodiment, the modified with at least one non - naturally encoded amino relaxin is recombinant human relaxin with one non - naturally acid . This specification will provide some embodiments , encoded amino acid . In yet another embodiment, the relaxin however it should be appreciated that these embodiments are is relaxin with more than one non -naturally encoded amino for the purpose of illustrating the invention , and are not to acid . In yet another embodiment of the present invention , the be construed as limiting the scope of the invention as defined 25 relaxin has a non - naturally encoded amino acid linked to a by the claims. water soluble polymer . In related embodiments , the relaxin In another aspect of the present invention , relaxin poly - may be administered daily , in an injectable formulation , as peptides with at least one non - naturally encoded amino acid a sustained release formulation , or as a continuous infusion . are attached to at least one water soluble polymer. In another aspect, the invention relates to the treatment of In one aspect , the invention relates to a method of 30 infections or ischemic wounds by administering a therapeu promoting angiogenesis in a mammal in need thereof by tically effective amount of relaxin . In a particularly preferred administering a therapeutically effective amount of relaxin . embodiment, the infection or ischemic wound is one where In another embodiment, relaxin is administered in an injury has resulted from lack of oxygen due to poor circu amount sufficient to maintain a serum concentration of at lation . least about 1 ng /ml . In a further embodiment the relaxin 35 In yet another aspect of the invention , there is provided a polypeptide is human relaxin (hR2 ) . method of using relaxin polypeptides of the present inven The present invention provides methods of treating indi - tion for the manufacture of a medicant for the treatment of viduals with diminished arterial compliance an effective an infection or ischemic wound , or for the manufacture of a amount of a formulation comprising a relaxin receptor medicant for the promotion of angiogenesis . In another agonist . In a preferred embodiment the relaxin receptor 40 aspect, the present invention relates to the treatment of agonist is a recombinant human relaxin , e . g . , human H2 osteodegenerative joint dysfunction , and in another aspect relaxin . the treatment of the osteodegenerative joint dysfunction In one embodiment of the invention , the invention pro comprises hR2 in addition to one or more adjuvants , includ vides a method of increasing arterial compliance in a sub ing but not limited to glucosamine . In another aspect , the ject, wherein said method comprises measuring global arte - 45 present invention relates to the treatment of alzheimer ' s rial compliance in said subject; determining that said global disease , and in another aspect the treatment of the alzheim arterial compliance is diminished in said subject relative to er ' s disease comprises hR2 in addition to one or more global arterial compliance in a healthy subject; and admin - adjuvants , including but not limited to estrogen . In another istering to said subject a pharmaceutical formulation com - embodiment, this invention relates to a method of modulat prising relaxin to increase arterial compliance in said sub - 50 ing the reproductive physiology of mammals comprising ject. Global arterial compliance may be measured , in one administering to the mammal a therapeutically effective embodiment , from the diastolic decay of the aortic pressure amount of the composition herein . waveform using the area method . In another embodiment, The invention further provides methods for treating global arterial compliance may be calculated as the stroke angiotensin - II ( AngII) -mediated vasoconstriction . These volume- to - pulse pressure ratio , where the stroke volume is 55 methods generally comprise administering a formulation defined as the ratio of cardiac output to heart rate . comprising an amount of relaxin effective to reverse, inhibit, In related embodiments , the local arterial compliance or or reduce the vasoconstricting effects of AngII. the regional arterial compliance of a subject may be mea - The invention further provides methods for treating sured in addition to or as an alternative to the global arterial endothelin - 1 (ET - 1 ) -mediated vasoconstriction . These meth compliance measurement and , if the local or regional arterial 60 ods generally comprise administering a formulation com compliance is diminished relative to the local or regional prising an amount of relaxin effective to reverse , inhibit , or arterial compliance expected for a similarly situated healthy reduce the vasoconstricting effects of ET- 1 . In some embodi individual, relaxin may be administered to increase arterial ments , the methods comprise increasing endothelin type B compliance in that individual. receptor activation in a cell in a blood vessel by adminis In further embodiments , the subject to whom relaxin is 65 tering relaxin to the individual. administered suffers from one or more of the following The invention further provides methods for treating an disorders : atherosclerosis , Type 1 diabetes, Type 2 diabetes, ischemic condition , generally comprising administering a US 9 ,962 ,450 B2 formulation comprising an amount of relaxin effective to comprising a poly ( ethylene glycol) moiety . In some embodi stimulate or promote angiogenesis and /or vasodilation , ments , at least one amino acid is a non -naturally encoded thereby treating the ischemic condition . The methods are amino acid . useful in treating a variety of ischemic conditions. In some In some embodiments , the relaxin polypeptide comprises embodiments , methods are provided for treating an ischemic 5 at least two amino acids linked to a water soluble polymer condition which arises as a result of myocardial infarct . In comprising a poly ( ethylene glycol) moiety . In some embodi other embodiments , methods are provided for treating an ments , at least one amino acid is a non -naturally encoded amino acid . ischemic condition associated with a wound . Thus, the In some embodiments, one or more non - naturally invention further provides methods for promoting wound* 10 encoded amino acids are incorporated in one or more of the healing. following positions in any of the relaxin or prorelaxin The invention further provides methods for stimulating polypeptides , relaxin analogs, prorelaxin , relaxin A chain , angiogenic and /or vasodilatory cytokine expression gener relaxin B chain Alal Asp relaxin , or relaxin polypeptides : in ally comprising administering a formulation comprising an the A chain before position 1 (i . e . at the N - terminus ), 1, 2 , amount of relaxin effective to vasodilate blood vessels 15 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , and / or stimulate or promote angiogenic cytokine production . 21 , 22 , 23 , 24 , 25 ( i .e . , at the carboxyl terminus of the In some embodiments , the methods provide for stimulating protein ), and any combination thereof (SEQ ID NO : 4 ) expression of basic fibroblast growth factor (bFGF ) and /or and /or in the B chain before position 1 (i . e . at the N - termi vascular endothelial cell growth factor (VEGF ). Such meth nus ) , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , ods are useful in treating a wide variety of diseases which 20 18 , 19 , 20, 21, 22 , 23 , 24 , 25 , 26 , 27, 28, 29, 30 (SEQ ID NO : can be treated by increasing blood flow at or near the site of 5 or the corresponding amino acids in SEQ ID NO : 6 ) . In disease . some embodiments , one or more non - naturally encoded The invention further provides a method of increasing amino acids are incorporated in one or more of the following renal vasodilation and hyperfiltration , generally comprising positions in any of the relaxin or prorelaxin polypeptides : in administering a formulation comprising an amount of 25 the A chain before position 1 ( i. e . at the N -terminus ) , 1, 2 , relaxin . These methods are useful in treating a variety of 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , renal pathologies. Accordingly , the invention further pro 21, 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , vides methods of treating a renal pathology related to 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , vasoconstriction . 53 , 54 ( i. e ., at the carboxyl terminus of the protein ) , and any The invention further provides a method of reducing 30 combination thereof (SEQ ID NO : 1 or the corresponding pulmonary hypertension , generally comprising administer amino acids in SEQ ID NO : 2 or 3 ) . In some embodiments, one or more non -naturally encoded amino acids are incor ing a formulation comprising an amount of relaxin . porated in one or more of the following positions in relaxin : In a patents assigned to Connetics Corporation and to before position 1 (i . e . at the N -terminus ), 1 , 2 , 3 , 4, 5 , 6 , 7 , BAS Medical, Inc ., U . S . Pat. Nos. 6 ,211 , 147 and 6 ,780 , 836° 35 8, 9, 10 , 11, 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22, 23 , 24 , respectively , both incorporated herein by reference ,methods 25 , 26 , 27 , 28 , 29, 30 , 31 , 32 , 33, 34 , 35 , 36 , 37 , 38 , 39 , 40 , of promoting angiogenesis using relaxin were disclosed . In 41, 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51, 52 , 53 , 54 , 55 , 56 , a patent assigned to Genentech , Inc ., U .S . Pat . No . 5 , 759, 57 , 58 , 59 , 60 , 61, 62 , 63 , 64 , 65 , 66 , 67, 68 , 69 , 70 , 71 , 72 , 807 , which is herein incorporated by reference, a process for 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81, 82 , 83 , 84 , 85 , 86 , 87 , 88 , prokaryotic production of relaxin from prorelaxin is dis - 40 89 ( i. e ., at the carboxyl terminus of the protein of SEQ ID closed . Yue U . S . Pat . No . 6 , 251, 863 discloses methods of NO : 3 ). treating osteodegenerative joint dysfunction and methods of In some embodiments a non -naturally encoded amino treating Alzheimer ' s by administering relaxin medicaments acid is incorporated in the A chain at amino acid position 1 further comprising glucosamine sulfate and estrogen , (SEQ ID NO : 4 ). In some embodiments a non - naturally respective for each of the conditions, and the specification of 45 encoded amino acid is incorporated in the A chain at amino this patent is also herein incorporated by reference in its acid position 5 ( SEQ ID NO : 4 ) . In some embodiments a entirety . non -naturally encoded amino acid is incorporated in the B In some embodiments , the relaxin polypeptide comprises chain at amino acid position 7 ( SEQ ID NO : 5 or SEQ ID one or more post - translational modifications . In some NO : 6 ) . In some embodiments a non - naturally encoded embodiments , the relaxin polypeptide is linked to a linker , 50 amino acid is incorporated in the A chain at amino acid polymer , or biologically active molecule . In some embodi position 2 ( SEQ ID NO : 4 ). In some embodiments a non ments , the relaxin polypeptide is linked to a bifunctional naturally encoded amino acid is incorporated in the A chain polymer , bifunctional linker, or at least one additional at amino acid position 13 (SEQ ID NO : 4 ) . In some embodi relaxin polypeptide . ments a non - naturally encoded amino acid is incorporated in In some embodiments , the non - naturally encoded amino 55 the B chain at amino acid position 5 (SEQ ID NO : 5 or SEQ acid is linked to a water soluble polymer . In some embodi - ID NO : 6 ) . In some embodiments a non -naturally encoded ments , the water soluble polymer comprises a poly ( ethylene amino acid is incorporated in the A chain at amino acid glycol) moiety . In some embodiments , the non -naturally position 18 . In some embodiments a non -naturally encoded encoded amino acid is linked to the water soluble polymer amino acid is incorporated in the B chain at amino acid with a linker or is bonded to the water soluble polymer . In 60 position 5 . In some embodiments a non - naturally encoded some embodiments , the poly ( ethylene glycol) molecule is a amino acid is incorporated in the B chain at amino acid bifunctional polymer . In some embodiments , the bifunc - position 28 . In some embodiments , one or more non tional polymer is linked to a second polypeptide . In some naturally encoded amino acids are incorporated in one of the embodiments , the second polypeptide is a relaxin polypep following positions in the relaxin polypeptides: in the A tide . 65 chain at amino acid position 1 , 2 , 5 , 13 , 18 (SEQ ID NO : 4 In some embodiments , the relaxin polypeptide comprises or the corresponding amino acids in SEQ ID NO : 1 , SEQ ID at least two amino acids linked to a water soluble polymer NO : 2 , SEQ ID NO : 3 , or other known relaxin sequences) . US 9 , 962 , 450 B2

In some embodiments , one or more non -naturally encoded (SEO ID NO : 1 or the corresponding amino acids in known amino acids are incorporated in one of the following posi relaxin sequences ) . In some embodiments , the non - naturally tions in the relaxin polypeptides : in the A chain at amino acid encoded amino acid at one or more of these positions is position 1, 2 , 5 , 13 (SEQ ID NO : 4 or the corresponding linked to a water soluble polymer , including but not limited amino acids in SEQ ID NO : 1 , SEQ ID NO : 2 , SEQ ID NO : 5 to : in the A chain 2 , 5 ( SEQ ID NO : 1 or the corresponding 3 , or other known relaxin sequences ) . In some embodiments , amino acids in known relaxin sequences ). In some embodi one or more non -naturally encoded amino acids are incor - ments , the non - naturally encoded amino acid at one or more porated in one of the following positions in the relaxin of these positions is linked to a water soluble polymer, polypeptides : in the A chain at amino acid position 1 , 2 , 5 including but not limited to : in the A chain 2 , 5 , 13 , 18 (SEQ ( SEQ ID NO : 4 or the corresponding amino acids in SEQ ID 10 ID NO : 1 or the corresponding amino acids in known relaxin NO : 1 , SEQ ID NO : 2 , SEQ ID NO : 3 , or other known sequences ) . In some embodiments , the non -naturally relaxin sequences ) . In some embodiments , one or more encoded amino acid at one or more of these positions is non - naturally encoded amino acids are incorporated in one linked to a water soluble polymer , including but not limited of the following positions in the relaxin polypeptides : in the to : in the B chain 5 , 7 (SEQ ID NO : 5 or 6 or the A chain at amino acid position 2 or 5 ( SEQ ID NO : 4 or the 15 corresponding amino acid positions in SEQ ID NO : 1, 2 , 3 ) . corresponding amino acids in SEQ ID NO : 1 , SEQ ID NO : In some embodiments , the non - naturally encoded amino 2 , SEQ ID NO : 3, or other known relaxin sequences ). acid at position 5 in the B chain (SEQ ID NO : 5 or 6 or the In some embodiments , a non -naturally encoded amino corresponding amino acid positions in SEQ ID NOs: 1, 2 , 3 ) acids is incorporated in one of the following positions in is linked to a water soluble polymer . In some embodiments , relaxin polypeptides : in the B chain at amino acid position 20 the non - naturally encoded amino acid at position 7 in the B 5 or 7 (SEQ ID NO : 5 or SEQ ID NO : 6 , or the correspond chain (SEQ ID NO : 5 or 6 or the corresponding amino acid ing amino acid positions in SEQ ID NOs: 1 , 2 , or 3 ) . In some positions in SEQ ID NOs: 1 , 2 , 3 ) is linked to a water soluble embodiments , a non -naturally encoded amino acid is incor - polymer porated at position 7 in the B chain ( SEQ ID NO : 5 or SEQ In some embodiments , one or more non -naturally ID NO : 6 , or the corresponding amino acid positions in SEQ 25 encoded amino acids are incorporated in one or more of the ID NOs: 1 , 2 , or 3 ) . In some embodiments, a non -naturally following positions in any of the relaxin or prorelaxin encoded amino acid is incorporated at position 5 in the B polypeptides: B chain positions 5 , 7 ( SEQ ID NO : 5 or 6 or chain (SEQ ID NO : 5 or SEQ ID NO : 6 , or the correspond the corresponding amino acid positions in SEQ ID NOs: 1 , ing amino acid positions in SEQ ID NOs: 1 , 2 , or 3 ) . In some 2 , 3 ) and A chain positions 1 , 5 , 2 , 13 , 18 (SEQ ID NO : 4 embodiments , a non - naturally encoded amino acid is incor - 30 or the corresponding amino acid positions in SEQ ID NOs: porated at position 7 in the B chain ( SEQ ID NO : 5 or SEQ 1 , 2 , 3 ) . In some embodiments , one or more non -naturally ID NO : 6 , or the corresponding amino acid positions in SEQ encoded amino acids are incorporated in one or more of the ID NOs: 1 , 2 , or 3 ) . following positions in any of the relaxin or prorelaxin In one embodiments , a non -naturally encoded amino polypeptides : B chain positions 5 , 7 (SEQ ID NO : 5 or 6 or acids is incorporated in one of the following positions in the 35 the corresponding amino acid positions in SEQ ID NOs: 1 , relaxin polypeptides: in the A chain at amino acid positions 2 , 3 ) and A chain positions 1 , 5 , 2 , 13 , 18 (SEQ ID NO : 4 1, 5 , 2 , 13 , 18 (SEQ ID NO : 4 or corresponding amino acid or the corresponding amino acid positions in SEQ ID NOs: positions in SEQ ID NO . 1 , 2 , 3 ) , in the B chain at amino 1 , 2 , 3 ) and the non - naturally encoded amino acid is linked acid positions 7 , 5 (SEQ ID NO : 5 or 6 , or corresponding to a water soluble polymer . amino acid positions in SEQ ID NO : 1 , 2 , 3 ) . In some 40 Methods of the present invention could be used to pro embodiments , one or more non -naturally encoded amino mote angiogenesis , promote vasodilation , promote non acids are incorporated in one of the following positions in hypotensive vasodilation , to treat hypertension , including the relaxin polypeptides : in the A chain at amino acid but not limited to renal hypertension , pulmonary hyperten positions 1 , 5 , 2 , 13 , 18 ( SEQ ID NO : 4 or corresponding sion , and cardiac hypertension ( U . S . Pat. Nos. 6 , 723 , 702 ; amino acid positions in SEQ ID NO . 1 , 2 , 3 ) , in the B chain 45 and 6 , 780 , 836 both hereby incorporated by reference in their at amino acid positions 7 , 5 (SEQ ID NO : 5 or 6 , or entirety ) , discloses formation and use of crystals of a relaxin corresponding amino acid positions in SEQ ID NO : 1 , 2 , 3 ) . analog . In some embodiments , the non - naturally encoded amino Formulations acid at one or more of these positions is linked to a water in the broad practice of the present invention , it also is soluble polymer , including but not limited to , positions: in 50 contemplated that a formulation may contain a mixture of the A chain before position 1 ( i . e . at the N -terminus ) , 1 , 2 , two or more of a relaxin , a relaxin dimer , a relaxin analog , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11, 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , an acylated relaxin , or acylated relaxin analog with at least 21 , 22 , 23 , 24 , 25 (i . e ., at the carboxyl terminus of the one of the components of the mixture containing a non protein ), and any combination thereof ( SEQ ID NO : 4 or the naturally encoded amino acid . In another embodiment of the corresponding amino acids in known relaxin sequences ) 55 present invention , the formulations containing a mixture of and / or in the B chain before position 1 ( i. e . at the N - termi two or more of relaxin , a relaxin analog , an acylated relaxin , nus ), 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , or acylated relaxin analog with at least one of the compo 18 , 19 , 20 , 21, 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29, 30 (SEQ ID NO : nents of the mixture containing a non - naturally encoded 5 or 6 or the corresponding amino acids in known relaxin amino acid also includes at least one water soluble polymer sequences ) . In some embodiments , the non -naturally 60 attached to at least one of the non - naturally encoded amino encoded amino acid at one or more of these positions is acids . linked to a water soluble polymer, including but not limited The present invention also includes heterogenous mix to : in the A chain 1, 2 , 5 , 13, 18 (SEQ ID NO : 1 or the tures wherein relaxin polypeptides and relaxin analogs are corresponding amino acids in known relaxin sequences ) . In prepared by the methods disclosed in this invention and are some embodiments , the non -naturally encoded amino acid at 65 then mixed so that a formulation may be administered to a one or more of these positions is linked to a water soluble patient in need thereof which contains , for example , 25 % polymer , including but not limited to : in the A chain 1 , 2, 5 relaxin polypeptide containing a non -naturally encoded US 9 , 962 , 450 B2 amino acid at position 28 of the B chain which has been tions , J . M . Harris , Plenus Press , New York (1992 ) , and in pegylated , 25 % relaxin polypeptide containing a non -natu Advanced Drug Reviews, 16 : 157 - 182 ( 1995 ) . rally encoded amino acid at position 10 of the B chain , said In one particular embodiment of the invention , the PEG non -naturally encoded amino acid coupled to a water soluble portion of the conjugate is absent one or more lipophilic polymer , and 50 % relaxin polypeptide wherein a non - 5 groups effective to significantly modify the water - soluble naturally encoded amino acid occurs at position 31 of the B nature of the polymer or of the polymer- relaxin conjugate . chain of relaxin (SEQ ID NO : 2 ; alternatively SEQ ID NOs: That is to say , the polymer or non - relaxin portion of a 4 , 6 , 8 , 10 , or 12 ) . All different mixtures of different conjugate of the invention may contain a group of atoms percentage amounts of relaxin polypeptide variants wherein considered to be more lipophilic than hydrophilic ( e . g . , a the relaxin polypeptides include a variety ( 1 ) with differently 10 carbon chain having from about 2 to 8 - 12 carbon atoms ) , sized PEGs, or ( 2 ) PEGs are included at different positions however, if the presence of such a group or groups is not in the sequence . This is intended as an example and should effective to significantly alter the hydrophilic nature of the in no way be construed as limiting to the formulations made polymer or of the conjugate , then such a moiety may be possible by the present invention and will be apparent to contained in the conjugates of the invention . That is to say, those of skill in the art . In an additional embodiment , the 15 through site - specific mutations of relaxin , relaxin polypep relaxin polypeptide variants to include in the formulation tides , and relaxin analogs , a relaxin conjugate of the inven mixture will be chosen by their varying dissociation times so tion itself may exhibit hydrophilic , rather than lipophilic or that the formulation may provide a sustained release of amphiphilic . In certain embodiments of the invention where relaxin for a patient in need thereof. a lipophilic moiety may be present, the moiety is preferably Formulations of the present invention may include a 20 not positioned at a terminus of a PEG chain . glucagon . Branched PEGs for use in the conjugates of the invention Other Embodiments of the Present Invention Including include those described in International Patent Publication Formulation for Inhalation WO 96 / 21469 , the contents of which is expressly incorpo In an additional embodiment of the present invention , it is rated herein by reference in its entirety . Generally , branched possible to use the technology disclosed herein for the 25 PEGs can be represented by the formula R ( PEG - OH ) .sub . n , production of relaxin analogs with increased pharmacoki- where R represents the central “ core ” molecule and . sub . n netic and pharmacodynamic properties for patient use via represents the number of arms. Branched PEGs have a administration to the lung, resulting in elevated blood levels central core from which extend 2 or more “ PEG ” arms. In of relaxin that are sustained for at least 6 hours , and more a branched configuration , the branched polymer core pos typically for at least 8 , 10 , 12 , 14 , 18 , 24 hours or greater 30 sesses a single reactive site for attachment to relaxin . post - administration . Another embodiment of the present Branched PEGs for use in the present invention will typi invention allows for advantageous mixtures of relaxin ana - cally comprise fewer than 4 PEG arms, and more preferably , logs suitable for therapeutic formulations designed to be will comprise fewer than 3 PEG arms. Branched PEGs offer administered to patients as an inhalant. the advantage of having a single reactive site , coupled with In some embodiments of the present invention , the fol- 35 a larger, more dense polymer cloud than their linear PEG lowing sites in the native relaxin molecule may be substi - counterparts . One particular type of branched PEG can be tuted with non - naturally encoded amino acids and optionally represented as (MeO -PEG - ) .sub . p R - X , where p equals 2 or further modified by covalent attachment of a water soluble 3 , R is a central core structure such as lysine or glycerol polymer, such as PEG : the 2 C - termini of the A and B chains, having 2 or 3 PEG arms attached thereto , and X represents Arg22B , His1OB , His5A , Glu4A , Glu17A , Glu13B , and 40 any suitable functional group that is or that can be activated Glu21B . for coupling to relaxin . One particularly preferred branched In addition to native relaxin , the present invention pro - PEG is mPEG2- NHS (Shearwater Corporation , Alabama ) vides for non - native relaxin polypeptides and relaxin ana - having the structure mPEG2 - lysine - succinimide . logs having one or more non - naturally encoded amino acids In yet another branched architecture, “ pendant PEG ” has substituted or inserted into the signal sequence thatmay also 45 reactive groups for protein coupling positioned along the provide a site for the incorporation of one or more water PEG backbone rather than at the end of PEG chains. The soluble polymers , such as PEG . This embodiment of the reactive groups extending from the PEG backbone for invention is particularly useful for introducing additional, coupling to relaxin may be the same or different. Pendant customized pegylation - sites within the relaxin molecule, for PEG structures may be useful but are generally less pre example , for forming a PEG -relaxin having improved resis - 50 ferred , particularly for compositions for inhalation . tance to enzymatic degradation . Such an approach provides Alternatively , the PEG - portion of a PEG - relaxin conju greater flexibility in the design of an optimized relaxin gate may possess a forked structure having a branched conjugate having the desired balance of activity , stability , moiety at one end of the polymer chain and two free reactive solubility , and pharmacological properties . Mutations can be groups ( or any multiple of 2 ) linked to the branched moiety carried out, i. e ., by site specific mutagenesis , at any number 55 for attachment to relaxin . Exemplary forked PEGs are of positions within the relaxin molecule. PEGs for use in the described in International Patent Publication No . WO present invention may possess a variety of structures : linear , 99 / 45964, the content of which is expressly incorporated forked , branched , dumbbell, and the like . Typically , PEG is herein by reference. The forked polyethylene glycol may activated with a suitable activating group appropriate for optionally include an alkyl or “ R ” group at the opposing end coupling a desired site or sites on the relaxin molecule . An 60 of the polymer chain . More specifically , a forked PEG activated PEG will possess a reactive group at a terminus for relaxin conjugate in accordance with the invention has the reaction with relaxin . Representative activated PEG deriva - formula : R - PEG - L ( Y -relaxin ) n where R is alkyl , L is a tives and methods for conjugating these agents to a drug hydrolytically stable branch point and Y is a linking group such as relaxin are known in the art and further described in that provides chemical linkage of the forked polymer to Zalipsky , S ., et al ., “ Use of Functionalized Poly (Ethylene 65 relaxin , and n is a multiple of 2 . L may represent a single Glycols ) for Modification of Polypeptides ” in Polyethylene “ core” group , such as “ _ CH — ” , or may comprise a longer Glycol Chemistry : Biotechnical and Biomedical Applica - chain of atoms . Exemplary L groups include lysine , glyc US 9 , 962 , 450 B2 12 erol, pentaerythritol, or sorbitol. Typically , the particular For instance, a PEG - cleavable linkage -relaxin conjugate branch atom within the branching moiety is carbon . should maintain its integrity upon manufacturing of the final In one particular embodiment of the invention , the linkage pharmaceutical composition , upon dissolution in an appro of the forked PEG to the relaxin molecule , ( Y ) , is hydro - priate delivery vehicle , if employed , and upon administra lytically stable . In a preferred embodiment , n is 2 . Suitable 5 tion irrespective of route . Y moieties , prior to conjugation with a reactive site on Thus, in another embodiment of the present invention , relaxin , include but are not limited to active esters , active one or more non -naturally encoded amino acids are incor carbonates , aldehydes, isocyanates, isothiocyanates, epox - porated into a single chain relaxin or single chain relaxin ides, alcohols , maleimides , vinylsulfones, hydrazides, dith - analog . iopyridines , and iodacetamides . Selection of a suitable acti - 10 In some embodiments , the polypeptide of the invention vating group will depend upon the intended site of comprises one or more non - naturally encoded amino acid attachment on the relaxin molecule and can be readily substitution , addition , or deletion in the signal sequence . In determined by one of skill in the art. The corresponding Y some embodiments , the polypeptide of the invention com group in the resulting PEG - relaxin conjugate is that which prises one or more non -naturally encoded amino acid sub results from reaction of the activated forked polymer with a 15 stitution , addition , or deletion in the signal sequence for suitable reactive site on relaxin . The specific identity of such relaxin or any of the relaxin analogs or polypeptides dis the final linkage will be apparent to one skilled in the art. For closed within this specification . In some embodiments , the example , if the reactive forked PEG contains an activated polypeptide of the invention comprises one or more natu ester , such as a succinimide or maleimide ester , conjugation rally encoded amino acid substitution , addition , or deletion via an amine site on relaxin will result in formation of the 20 in the signal sequence as well as one or more non - naturally corresponding amide linkage . These particular forked poly encoded amino acid substitutions , additions, or deletions in mers are particularly attractive since they provide conjugates the signal sequence for relaxin or any of the relaxin analogs having a molar ratio of relaxin to PEG of 2 : 1 or greater. Such or polypeptides disclosed within this specification . In some conjugates may be less likely to block the relaxin receptor embodiments , one or more non - natural amino acids are site , while still providing the flexibility in design to protect 25 incorporated in the leader or signal sequence for relaxin or the relaxin against enzymatic degradation , e . g . , by relaxin any of the relaxin analogs or polypeptides disclosed within degrading enzyme. this specification. In a related embodiment, a forked PEG -relaxin conjugate In some embodiments , the relaxin polypeptide comprises may be used in the present invention , represented by the a substitution , addition or deletion that modulates affinity of formula : R - [PEG - L ( Y -relaxin ) 2 ] n . In this instance R repre - 30 the relaxin polypeptide for a relaxin polypeptide receptor or sents a non -naturally encoded amino acid having attached binding partner , including but not limited to , a protein , thereto at least one PEG - di- relaxin conjugate . Specifically, polypeptide , small molecule , or nucleic acid . In some preferred forked polymers in accordance with this aspect of embodiments , the relaxin polypeptide comprises a substitu the invention are those were n is selected from the group tion , addition , or deletion that increases the stability of the consisting of 1 , 2 , 3 , 4 , 5 , and 6 . In an alternative embodi- 35 relaxin polypeptide when compared with the stability of the ment, the chemical linkage between the non -natural amino corresponding relaxin without the substitution , addition , or acid within relaxin , relaxin polypeptide , or relaxin analog deletion . Stability and/ or solubility may be measured using and the polymer branch point may be degradable ( i . e . , a number of different assays known to those of ordinary skill hydrolytically unstable ) . Alternatively , one or more degrad in the art . Such assays include but are not limited to able linkages may be contained in the polymer backbone to 40 SE -HPLC and RP -HPLC . In some embodiments , the relaxin allow generation in vivo of a PEG - relaxin conjugate having polypeptide comprises a substitution , addition , or deletion a smaller PEG chain than in the initially administered that modulates the immunogenicity of the relaxin polypep conjugate . For example , a large and relatively inert conju - tide when compared with the immunogenicity of the corre gate ( i. e ., having one or more high molecular weight PEG sponding relaxin without the substitution , addition , or dele chains attached thereto , e . g ., one or more PEG chains having 45 tion . In some embodiments , the relaxin polypeptide a molecular weight greater than about 10 , 000 , wherein the comprises a substitution , addition , or deletion that modu conjugate possesses essentially no bioactivity ) may be lates serum half- life or circulation time of the relaxin administered , which then either in the lung or in the blood - polypeptide when compared with the serum half - life or stream , is hydrolyzed to generate a bioactive conjugate circulation time of the corresponding relaxin without the possessing a portion of the originally present PEG chain . 50 substitution , addition , or deletion . Upon in - vivo cleavage of the hydrolytically degradable In some embodiments , the relaxin polypeptide comprises linkage , either free relaxin (depending upon the position of a substitution , addition , or deletion that increases the aque the degradable linkage ) or relaxin having a small polyeth - ous solubility of the relaxin polypeptide when compared to ylene tag attached thereto , is then released and more readily aqueous solubility of the corresponding relaxin without the absorbed through the lung and / or circulated in the blood . 55 substitution , addition , or deletion . In some embodiments , the In one feature of this embodiment of the invention , the relaxin polypeptide comprises a substitution , addition , or intact polymer - conjugate , prior to hydrolysis , is minimally deletion that increases the solubility of the relaxin polypep degraded upon administration , such that hydrolysis of the tide produced in a host cell when compared to the solubility cleavable bond is effective to govern the slow rate of release of the corresponding relaxin without the substitution , addi of active relaxin into the bloodstream , as opposed to enzy - 60 tion , or deletion . In some embodiments, the relaxin poly matic degradation of relaxin prior to its release into the peptide comprises a substitution , addition , or deletion that systemic circulation . increases the expression of the relaxin polypeptide in a host Appropriate physiologically cleavable linkages include cell or increases synthesis in vitro when compared to the but are not limited to ester, carbonate ester, carbamate , expression or synthesis of the corresponding relaxin without sulfate , phosphate , acyloxyalkyl ether, acetal, and ketal. 65 the substitution , addition , or deletion . The relaxin polypep Such conjugates should possess a physiologically cleavable tide comprising this substitution retains agonist activity and bond that is stable upon storage and upon administration . retains or improves expression levels in a host cell . In some US 9 , 962 , 450 B2 13 14 embodiments , the relaxin polypeptide comprises a substitu wherein n is 0 - 10 ; R1 is an alkyl, aryl, substituted alkyl, tion , addition , or deletion that increases protease resistance or substituted aryl; R2 is H , an alkyl, aryl, substituted alkyl, of the relaxin polypeptide when compared to the protease and substituted aryl; and R3 is H , an amino acid , a poly resistance of the corresponding relaxin without the substi tution , addition , or deletion . In some embodiments , the 5 peptidei , or an amino terminus modification group , and R4 is relaxin polypeptide comprises a substitution , addition , or 5 H , an amino acid , a polypeptide , or a carboxy terminus deletion that modulates signal transduction activity of the modification group . relaxin receptor when compared with the activity of the In some embodiments , the non -naturally encoded amino receptor upon interaction with the corresponding relaxin acid comprises an aminooxy group . In some embodiments , polypeptide without the substitution , addition , or deletion . In o the non - naturally encoded amino acid comprises a hydrazide some embodiments , the relaxin polypeptide comprises a group . In some embodiments , the non -naturally encoded substitution , addition , or deletion that modulates its binding amino acid comprises a hydrazine group . In some embodi to anothermolecule such as a receptor when compared to the ments , the non - naturally encoded amino acid residue com binding of the corresponding relaxin polypeptide without the prises a semicarbazide group . substitution , addition , or deletion . In some embodiments , the In some embodiments , the non -naturally encoded amino relaxin polypeptide comprises a substitution , addition , or 13 acid residue comprises an azide group . In some embodi deletion thatmodulates its anti - viral activity compared to the ments , the non - naturally encoded amino acid has the struc anti - viral activity of the corresponding relaxin polypeptide ture : without the substitution , addition , or deletion . In some embodiments , the relaxin polypeptide comprises a substitu tion , addition , or deletion that enhances its glucose metabo - 20 lizing activity compared to the glucose metabolizing activity ( CH2) ?R X (CH2 ) mN3 of the corresponding relaxin polypeptide without the sub stitution , addition , or deletion . R2HN COR3 In some embodiments , the relaxin polypeptide comprises a substitution , addition , or deletion that increases compat - 25 wherein n is 0 - 10 : R1 is an alkyl. arvl. substituted alkyl. ibility of the relax?n polypeptide with pharmaceutical pre - substituted arvl or not present: X is O . N . S or not present: servatives (e . g. , m -cresol , phenol, benzyl alcohol) when compared to compatibility of the corresponding relaxin m is 0 - 10 ; R2 is H , an amino acid , a polypeptide , or an without the substitution , addition , or deletion . This increased amino terminus modification group , and R3 is H , an amino compatibility would enable the preparation of a preserved 30 acid , a polypeptide , or a carboxy terminus modification pharmaceutical formulation that maintains the physiochemi cal properties and biological activity of the protein during In some embodiments , the non -naturally encoded amino storage . 8 acid comprises an alkyne group . In some embodiments , the In some embodiments , one or more engineered bonds are non - naturally encoded amino acid has the structure: created with one or more non -natural amino acids. The 35 intramolecular bond may be created in many ways , includ (CH2 ) ,R2X (CH2 ) mCCH ing but not limited to , a reaction between two amino acids in the protein under suitable conditions ( one or both amino acids may be a non -natural amino acid ) ; a reaction with two R2HN COR ; amino acids, each of which may be naturally encoded or 40 non - naturally encoded , with a linker , polymer, or other wherein n is 0 - 10 ; R1 is an alkyl, aryl, substituted alkyl, or molecule under suitable conditions; etc . substituted aryl; X is O , N , S or not present; m is 0 - 10 , R2 In some embodiments , one or more amino acid substitu is H , an amino acid , a polypeptide, or an amino terminus tions in the relaxin polypeptide may be with one or more modification group , and R3 is H . an amino acid , a polypep naturally occurring or non - naturally encoded amino acids. In 45 tide , or a carboxy terminus modification group . some embodiments the amino acid substitutions in the In some embodiments , the polypeptide is a relaxin poly relaxin polypeptide may be with naturally occurring or peptide agonist , partial agonist , antagonist, partial antago non - naturally encoded amino acids, provided that at least nist, or inverse agonist . In some embodiments , the relaxin one substitution is with a non - naturally encoded amino acid . polypeptide agonist, partial agonist, antagonist , partial In some embodiments , one or more amino acid substitutions 50 antagonist , or inverse agonist comprises a non -naturally in the relaxin polypeptide may be with one or more naturally encoded amino acid linked to a water soluble polymer. In occurring amino acids, and additionally at least one substi - some embodiments , the water soluble polymer comprises a tution is with a non -naturally encoded amino acid . poly ( ethylene glycol) moiety . In some embodiments , the In some embodiments , the non - naturally encoded amino relaxin polypeptide agonist , partial agonist , antagonist , par acid comprises a carbonyl group , an acetyl group , an ami- 55 tial antagonist, or inverse agonist comprises a non - naturally nooxy group , a hydrazine group , a hydrazide group , a encoded amino acid and one or more post -translational semicarbazide group , an azide group , or an alkyne group . modification , linker, polymer, or biologically active mol In some embodiments , the non - naturally encoded amino ecule . acid comprises a carbonyl group . In some embodiments , the The present invention also provides isolated nucleic acids non - naturally encoded amino acid has the structure : 60 comprising a polynucleotide that hybridizes under stringent conditions nucleic acids that encode relaxin polypeptides of SEQ ID NOs: 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , and 12 . The (CH2 ) , R COR2 present invention also provides isolated nucleic acids com prising a polynucleotide that hybridizes under stringent R3HN COR4 65 conditions nucleic acids that encode relaxin polypeptides of SEQ ID NOs: 1 and 2 . The present invention also provides isolated nucleic acids comprising a polynucleotide or poly US 9 , 962 , 450 B2 15 16 nucleotides that hybridize under stringent conditions to moiety . In some embodiments , the azide or alkyne group is polynucleotides that encode polypeptides shown as SEQ ID linked to the poly (ethylene glycol) molecule through an NOs: 1, 2 , 3 , 4 , 5, 6 , 7, 8 , 9 , 10 , 11, and 12 wherein the amide linkage . polynucleotide comprises at least one selector codon . The In some embodiments , the relaxin polypeptide linked to present invention also provides isolated nucleic acids com - 5 the water soluble polymer is made by reacting a relaxin prising a polynucleotide or polynucleotides that hybridize polypeptide comprising an azide -containing amino acid with under stringent conditions to polynucleotides that encode a poly ( ethylene glycol) molecule comprising an alkyne polypeptides shown as SEQ ID NOs: 1 and 2 wherein the moiety . In some embodiments , the azide or alkyne group is polynucleotide comprises at least one selector codon . The linked to the poly ( ethylene glycol) molecule through an present invention provides isolated nucleic acids comprising " amide linkage . a polynucleotide that encodes the polypeptides shown as In some embodiments , the poly ( ethylene glycol) molecule SEQ ID NOs. : 1 and 2 . The present invention also provides has a molecular weight of between about 0 . 1 kDa and about isolated nucleic acids comprising a polynucleotide that 100 kDa . In some embodiments, the poly ( ethylene glycol) encodes the polypeptides shown as SEQ ID NOs. : 1 , 2 , 3 , 4 , 16 molecule has a molecular weight of between 0 . 1 kDa and 50 5 , 6 , 7 , 8 , 9 , 10 , 11, and 12 . The present invention provides kDa . isolated nucleic acids comprising a polynucleotide that In some embodiments , the poly ( ethylene glycol) molecule encodes the polypeptides shown as SEQ ID NOs. : 1 and 2 is a branched polymer. In some embodiments , each branch with one or more non -naturally encoded amino acids. The of the poly (ethylene glycol) branched polymer has a present invention also provides isolated nucleic acids com - 20 molecular weight of between 1 kDa and 100 kDa , or prising a polynucleotide that encodes the polypeptides between 1 kDa and 50 kDa. shown as SEQ ID NOs .: 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , and In some embodiments , the water soluble polymer linked 12 with one or more non -naturally encoded amino acids. It to the relaxin polypeptide comprises a polyalkylene glycol is readily apparent to those of ordinary skill in the art that a moiety . In some embodiments , the non -naturally encoded number of different polynucleotides can encode any poly - 25 amino acid residue incorporated into the relaxin polypeptide peptide of the present invention . comprises a carbonyl group , an aminooxy group , a hydraz In some embodiments , the selector codon is selected from ide group , a hydrazine, a semicarbazide group, an azide the group consisting of an amber codon , ochre codon , opal group , or an alkyne group . In some embodiments , the codon , a unique codon , a rare codon , a five -base codon , and non -naturally encoded amino acid residue incorporated into a four -base codon . 30 the relaxin polypeptide comprises a carbonyl moiety and the The present invention also provides methods of making a water soluble polymer comprises an aminooxy , hydrazide , relaxin polypeptide linked to a water soluble polymer . In hydrazine, or semicarbazide moiety . In some embodiments , some embodiments , the method comprises contacting an the non - naturally encoded amino acid residue incorporated isolated relaxin polypeptide comprising a non -naturally 25 into the relaxin polypeptide comprises an alkyne moiety and encoded amino acid with a water soluble polymer compris - the water soluble polymer comprises an azide moiety . In ing a moiety that reacts with the non - naturally encoded some embodiments , the non - naturally encoded amino acid amino acid . In some embodiments , the non -naturally residue incorporated into the relaxin polypeptide comprises encoded amino acid incorporated into the relaxin polypep - an azidemoiety and the water soluble polymer comprises an tide is reactive toward a water soluble polymer that is 40 alkyne moiety . otherwise unreactive toward any of the 20 common amino The present invention also provides compositions com acids . In some embodiments , the non - naturally encoded prising a relaxin polypeptide comprising a non - naturally amino acid incorporated into the relaxin polypeptide is encoded amino acid and a pharmaceutically acceptable reactive toward a linker, polymer , or biologically active carrier . In some embodiments , the non -naturally encoded molecule that is otherwise unreactive toward any of the 20 45 amino acid is linked to a water soluble polymer . common amino acids. The present invention also provides cells comprising a In some embodiments , the relaxin polypeptide linked to polynucleotide encoding the relaxin polypeptide comprising the water soluble polymer is made by reacting a relaxin a selector codon . In some embodiments , the cells comprise polypeptide comprising a carbonyl- containing amino acid an orthogonal RNA synthetase and /or an orthogonal tRNA with a poly ( ethylene glycol) molecule comprising an ami- 50 for substituting a non -naturally encoded amino acid into the nooxy , hydrazine, hydrazide or semicarbazide group . In relaxin polypeptide. some embodiments , the aminooxy , hydrazine , hydrazide or The present invention also provides methods ofmaking a semicarbazide group is linked to the poly (ethylene glycol) relaxin polypeptide comprising a non -naturally encoded molecule through an amide linkage . In some embodiments , amino acid . In some embodiments , the methods comprise the aminooxy, hydrazine , hydrazide or semicarbazide group 55 culturing cells comprising a polynucleotide or polynucle is linked to the poly ( ethylene glycol) molecule through a otides encoding a relaxin polypeptide , an orthogonal RNA carbamate linkage . synthetase and /or an orthogonal tRNA under conditions to In some embodiments , the relaxin polypeptide linked to permit expression of the relaxin polypeptide ; and purifying the water soluble polymer is made by reacting a poly the relaxin polypeptide from the cells and/ or culture ( ethylene glycol) molecule comprising a carbonyl group 60 medium . with a polypeptide comprising a non -naturally encoded The present invention also provides methods of increasing amino acid that comprises an aminooxy, hydrazine , hydraz - therapeutic half -life , serum half - life or circulation time of ide or semicarbazide group . relaxin polypeptides . The present invention also provides In some embodiments , the relaxin polypeptide linked to methods of modulating immunogenicity of relaxin polypep the water soluble polymer is made by reacting a relaxin 65 tides . In some embodiments , the methods comprise substi polypeptide comprising an alkyne - containing amino acid tuting a non -naturally encoded amino acid for any one or with a poly ( ethylene glycol) molecule comprising an azide more amino acids in naturally occurring relaxin polypep US 9 , 962, 450 B2 17 18 tides and /or linking the relaxin polypeptide to a linker, a embodiments , the polypeptide is monoPEGylated . The pres polymer , a water soluble polymer, or a biologically active ent invention also provides a relaxin polypeptide comprising molecule. a linker , polymer, or biologically active molecule that is The present invention also provides methods of treating a attached to one or more non -naturally encoded amino acid patient in need of such treatment with an effective amount of 5 wherein said non -naturally encoded amino acid is riboso a relaxin molecule of the present invention . In some embodi mally incorporated into the polypeptide at pre - selected sites. ments, the methods comprise administering to the patient a therapeutically - effective amount of a pharmaceutical com Included within the scope of this invention is the relaxin position comprising a relaxin polypeptide comprising a leader or signal sequence an example of which can be seen non -naturally -encoded amino acid and a pharmaceutically 10 as proinulin . The heterologous leader or signal sequence acceptable carrier . In some embodiments , the non -naturally selected should be one that is recognized and processed , e . g . encoded amino acid is linked to a water soluble polymer . In by host cell secretion system to secrete and possibly cleaved some embodiments , the relaxin polypeptide is glycosylated . by a signal peptidase , by the host cell. A method of treating In some embodiments , the relaxin polypeptide is not gly a condition or disorder with relaxin or a relaxin polypeptide cosylated . or analog of the present invention is meant to imply treating The present invention also provides relaxin polypeptides with relaxin with or without a signal or leader peptide . comprising a sequence shown in SEQ ID NO : 1 and 2 , or The present invention also provides methods of inducing any other relaxin polypeptide sequence ( a non - limiting an increase in glucose metabolism , said method comprising example of these would be SEQ ID NOs: 3 through 12 ) administering relaxin to said cells in an amount effective to except that at least one amino acid is substituted by a 20 induce an increase in glucose metabolic activity . non -naturally encoded amino acid . In some embodiments , In another embodiment, conjugation of the relaxin poly the non - naturally encoded amino acid is linked to a water peptide comprising one or more non -naturally encoded soluble polymer. In some embodiments , the water soluble amino acids to another molecule , including but not limited polymer comprises a poly (ethylene glycol) moiety . In some to PEG , provides substantially purified relaxin due to the embodiments , the non - naturally encoded amino acid com - 25 unique chemical reaction utilized for conjugation to the prises a carbonyl group , an aminooxy group , a hydrazide non - natural amino acid . Conjugation of relaxin comprising group , a hydrazine group , a semicarbazide group , an azide one or more non - naturally encoded amino acids to another group , or an alkyne group . molecule , such as PEG , may be performed with other The present invention also provides pharmaceutical com - purification techniques performed prior to or following the positions comprising a pharmaceutically acceptable carrier 30 conjugation step to provide substantially pure relaxin . and a relaxin polypeptide comprising the sequence shown in SEQ ID NOs: 1 through 12 , or any other relaxin polypeptide BRIEF DESCRIPTION OF THE SEVERAL sequence , wherein at least one amino acid is substituted by VIEWS OF THE DRAWINGS a non - naturally encoded amino acid . The present invention also provides pharmaceutical compositions comprising a 35 FIG . 1 is a model of the crystal structure of relaxin are pharmaceutically acceptable carrier and a relaxin polypep - shown along with some amino acid residue positions tide comprising an A and B chain ( e . g . SEQ ID NO : 1 , 2 , and selected for substitution . 3 ; SEQ ID NOs: 4 and 5 or 4 and 6 would make relaxin , FIG . 2 is a model of the crystal structure of relaxin are etc . ) , or any other relaxin polypeptide sequence , wherein at shown along with some amino acid residue positions least one amino acid is substituted by a non - naturally 40 selected for substitution . encoded amino acid . The present invention also provides FIG . 3 is a model of the crystal structure of relaxin are pharmaceutical compositions comprising a pharmaceuti - shown along with some amino acid residue positions cally acceptable carrier and a relaxin polypeptide compris - selected for substitution . ing the sequence shown in SEQ ID NO : 1 , 2 , and /or 3 . The FIG . 4 is a drawing of the structure of the A ( SEQ ID NO : present invention also provides pharmaceutical composi - 45 4 ) and B (SEQ ID NO : 5 ) chain of human relaxin . tions comprising a pharmaceutically acceptable carrier and FIG . 5 shows an SDS - PAGE gel of the prorelaxin pro a relaxin polypeptide comprising the sequence shown in duced by these methods with a chain B1 amino acid as Ala SEQ ID NO : 1 - 3 . In some embodiments , the non -naturally and a para - acetyl phenylalanine in the 13th amino acid encoded amino acid comprises a saccharide moiety. In some position of the A chain , substituted for valine . embodiments , the water soluble polymer is linked to the 50 FIG . 6A shows an SDS - PAGE gel of unPEGylated relaxin polypeptide via a saccharide moiety . In some embodiments , V13pAF alongside a molecular weight marker in lane 1 and a linker , polymer, or biologically active molecule is linked recombinant and non -recombinant WT relaxin in lanes 3 and to the relaxin polypeptide via a saccharide moiety . 7 (non - reduced (NR ) and reduced ( R ) ). FIG . 6B shows an The present invention also provides a relaxin polypeptide SDS -PAGE gel of PEGylated relaxin V13pAF in lanes 3 and comprising a water soluble polymer linked by a covalent 55 4 (non - reduced (NR ) and reduced ( R ) ) alongside a molecu bond to the relaxin polypeptide at a single amino acid . In lar weight marker in lane 1 . some embodiments , the water soluble polymer comprises a FIG . 7 shows a graph of SD rat serum relaxin concentra poly ( ethylene glycol) moiety . In some embodiments , the tion in ng/ mL over time for differently PEGylated AV13 and amino acid covalently linked to the water soluble polymer is wild type relaxin polypeptides . a non - naturally encoded amino acid present in the polypep - 60 FIG . 8A shows a graph of the comparison of group mean tide. serum concentration versus time for all PEG -RLX groups The present invention provides a relaxin polypeptide dosed subcutaneously in Example 40 . A single dose injec comprising at least one linker , polymer, or biologically tion was administered to each animal. N = 5 animals per active molecule, wherein said linker, polymer, or biologi- group . Symbols indicate mean + SD of grouped serum con cally active molecule is attached to the polypeptide through 65 centrations versus time. a functional group of a non - naturally encoded amino acid FIG . 8B shows a graph of the individual animal serum ribosomally incorporated into the polypeptide . In some concentration time curves for SD rats dosed subcutaneously US 9 , 962 ,450 B2 19 20 with 0 . 5 mg/ kg of 20KPEG -AQ1 - RLX . A single , subcuta FIG . 17B shows a graph of the mean PEG - Relaxin neous dose was administered to each animal. N = 5 animals Clearance versus dose ; error bars = SD . per group . FIG . 18A shows a graph of the mean PEG -Relaxin FIG . 9A shows a graph of the individual animal serum Volume of distribution versus Dose ; error bars = SD . FIG . concentration time curves for SD rats dosed subcutaneously 5 18B shows a graph of a comparison of Serum - time concen with 0 . 5 mg/kg of PEG20 -AA5 -RLX . A single , subcutane - tration after a single intravenous or subcutaneous injection ous dose was administered to each animal. N = 5 animals per of 0 . 25 mg/ kg of 20KPEG -AQ1 Relaxin . group . FIGS . 19A - F show data from Phase I of Example 43. FIG . 19A shows the effect of IV infusion with wild - type relaxin FIG . 9B shows a graph of the individual animalcutaneously serum 10 on water intake and urine output. FIG . 19B shows a baseline concentration time curves for SD rats dosed subcutaneously for each group of Long - Evans rats from - 16 ours to 0 of with 0 . 5 mg/ kg of PEG20 - AR18 -RLX . A single , subcuta Phase I for urine output. FIG . 19C shows the effect of IV neous dose was administered to each animal. N = 5 animals infusion with wild -type relaxin on hematocrit . FIG . 19D per group . shows the effect of IV infusion with wild - type relaxin on FIG . 10A shows a graph of the individual animal serum 15 plasma BUN in the female Long- Evans rats . FIG . 19E concentration time curves for SD rats dosed intravenously shows water intake for each group of Long - Evans rats from with 0 . 5 mg/ kg of PEG20 -BV7 - RLX . A single , intravenous 0 to 6 hours of Phase I. FIG . 19F shows urine output for each dose was administered to each animal. N = 5 animals per group of Long - Evans rats from 0 to 6 hours of Phase I . group . FIGS. 20A - I show data from Phase II following admin FIG . 10B shows a graph of the individual animal serum 20 istration of vehicle ( control group ) and test groups with concentration time curves for SD rats dosed intravenously 0 . 1x , 0 . 3x and 1x administration of a 20K PEG -Relaxin with 0 . 5 mg/ kg of PEG20 -BW28 -RLX . A single , intrave variant with A chain substitution in position 1 with PAF nous dose was administered to each animal. N = 5 animals per bound to PEG from Example 43 . FIG . 20A shows the effect group . of on water intake and urine output. FIG . 20B shows the FIG . 11 shows a graph of the individual animal serum 25 effect on plasma sodium levels for each group of Long concentration time curves for SD rats dosed intravenously Evans rats following injection . FIG . 20C shows the effect on with 0 . 5 mg/ kg of PEG20 - AV13 -RLX . A single , intravenous plasma sodium change levels for each group of Long - Evans dose was administered to each animal. N = 5 animals per rats following injection . FIG . 20D shows the effect of group . PEG -Relaxin on plasma osmolarity . FIG . 20E shows the FIG . 12A shows a graph of a comparison of group mean 30 effect of IV infusion with PEG -Relaxin on plasma osmolar serum concentration versus time for wt rhRelaxin dosed ity change. FIG . 20F shows the effect of PEG - Relaxin subcutaneously . A single dose injection was administered to administration on BUN levels. FIG . 20G shows the effect of each animal. N = 5 animals per group . PEG -relaxin administration on water intake for each group FIG . 12B shows a graph of individual animal serum of Long - Evans rats from 0 to 6 hours of Phase II . FIG . 20H concentration time curves for SD rats dosed intravenously 35 shows baseline urine output for each group of Long -Evans with 0 . 5 mg/ kg of wt rhRelaxin . A single , intravenous dose rats from - 16 to 0 hours of Phase II . FIG . 201 shows the was administered to each animal. N = 5 animals per group . effect of PEG - relaxin administration on urine output for FIG . 13A shows a comparison of group mean serum each group of Long -Evans rats from 0 to 6 hours of Phase concentration versus time for all PEG - RLX groups dosed II . subcutaneously or intravenously . A single dose injection was 40 administered to each animal. N = 3 - 5 animals per group . DEFINITIONS FIG . 13B shows a graph of individual animal serum concentration time curves for SD rats dosed intravenously I t is to be understood that this invention is not limited to with 0 . 25 mg/ kg of 20KPEG -AQ1 - RLX . A single , intrave - the particular methodology , protocols , cell lines , constructs , nous dose was administered to each animal. N = 4 animals per 45 and reagents described herein and as such may vary . It is also group . to be understood that the terminology used herein is for the FIG . 14A shows a graph of individual animal serum purpose of describing particular embodiments only , and is concentration time curves for SD rats dosed subcutaneously not intended to limit the scope of the present invention , with 0 . 5 mg/ kg of PEG20 - AQ1- RLX . A single , subcutane - which will be limited only by the appended claims. ous dose was administered to each animal. N = 5 animals per 50 As used herein and in the appended claims, the singular group . forms “ a , " " an ,” and “ the” include plural reference unless FIG . 14B shows a graph of individual animal serum the context clearly indicates otherwise. Thus, for example , concentration time curves for SD rats dosed subcutaneously reference to a “ relaxin ” or “ relaxin polypeptide ” and various with 0 .25 mg/ kg of PEG20 - AQ1- RLX . A single , subcuta - hyphenated and unhyphenated forms is a reference to one or neous dose was administered to each animal. N = 3 animals 55 more such proteins and includes equivalents thereof known per group . to those of ordinary skill in the art , and so forth . FIG . 15 shows a graph of individual animal serum con Unless defined otherwise , all technical and scientific centration time curves for SD rats dosed intravenously with terms used herein have the same meaning as commonly 0 . 125 mg/ kg of PEG20 -AQ1 - RLX . A single , intravenous understood to one of ordinary skill in the art to which this dose was administered to each animal. N = 5 animals per 60 invention belongs . Although any methods, devices , and group . materials similar or equivalent to those described herein can FIG . 16A shows a graph of the mean PEG -Relaxin be used in the practice or testing of the invention , the Terminal Half - life versus dose; error bars = SD . FIG . 16B preferred methods, devices and materials are now described . shows a graph of mean PEG -Relaxin AUC ; nf versus dose ; All publications and patents mentioned herein are incor error bars = SD . 65 porated herein by reference for the purpose of describing FIG . 17A shows a graph of the mean PEG -Relaxin Cmax and disclosing , for example , the constructs and methodolo versus dose ; error bars = SD . gies that are described in the publications , which might be US 9 , 962 , 450 B2 21 22 used in connection with the presently described invention . 2 -mercaptoethanol , dithioerythritol, cysteine, cysteamine The publications discussed herein are provided solely for (2 -aminoethanethiol ) , and reduced glutathione . It is readily their disclosure prior to the filing date of the present appli apparent to those of ordinary skill in the art that a wide cation . Nothing herein is to be construed as an admission variety of reducing agents are suitable for use in the methods that the inventors are not entitled to antedate such disclosure 5 and compositions of the present invention . by virtue of prior invention or for any other reason . " Oxidizing agent ," as used hereinwith respect to protein The term " substantially purified ” refers to a relaxin poly - refolding , is defined as any compound or material which is peptide that may be substantially or essentially free of capable of removing an electron from a compound being components that normally accompany or interact with the oxidized . Suitable oxidizing agents include , but are not protein as found in its naturally occurring environment, i . e . 10 limited to , oxidized glutathione , cystine, cystamine , oxi a native cell, or host cell in the case of recombinantly dized dithiothreitol, oxidized erythreitol, and oxygen . It is produced relaxin polypeptides . Relaxin polypeptide that readily apparent to those of ordinary skill in the art that a may be substantially free of cellular material includes prepa - wide variety of oxidizing agents are suitable for use in the rations of protein having less than about 30 % , less than methods of the present invention . about 25 % , less than about 20 % , less than about 15 % , less 15 “ Denaturing agent” or “ denaturant, " as used herein , is than about 10 % , less than about 5 % , less than about 4 % , less defined as any compound or material which will cause a than about 3 % , less than about 2 % , or less than about 1 % (by reversible unfolding of a protein . The strength of a dena dry weight ) of contaminating protein . When the relaxin turing agent or denaturant will be determined both by the polypeptide or variant thereof is recombinantly produced by properties and the concentration of the particular denaturing the host cells , the protein may be present at about 30 % , 20 agent or denaturant. Suitable denaturing agents or denatur about 25 % , about 20 % , about 15 % , about 10 % , about 5 % , ants may be chaotropes, detergents , organic solvents , water about 4 % , about 3 % , about 2 % , or about 1 % or less of the miscible solvents , phospholipids, or a combination of two or dry weight of the cells . When the relaxin polypeptide or more such agents . Suitable chaotropes include, but are not variant thereof is recombinantly produced by the host cells , limited to , urea , guanidine , and sodium thiocyanate . Useful the protein may be present in the culture medium at about 5 25 detergents may include , but are not limited to , strong deter g / L , about 4 g / L , about 3 g / L , about 2 g / L , about 1 g / L , about gents such as sodium dodecyl sulfate, or polyoxyethylene 750 mg/ L , about 500 mg/ L , about 250 mg/ L , about 100 ethers (e . g . Tween or Triton detergents ) , Sarkosyl, mild mg/ L , about 50 mg/ L , about 10 mg/ L , or about 1 mg/ L or non - ionic detergents ( e . g ., digitonin ) , mild cationic deter less of the dry weight of the cells . Thus , " substantially gents such as N - > 2 , 3 - (Dioleyoxy ) - propyl - N , N , N - trimethyl purified " relaxin polypeptide as produced by the methods of 30 ammonium , mild ionic detergents (e . g . sodium cholate or the present invention may have a purity level of at least sodium deoxycholate ) or zwitterionic detergents including , about 30 % , at least about 35 % , at least about 40 % , at least but not limited to , sulfobetaines (Zwittergent ) , 3 - ( 3 - chlo about 45 % , at least about 50 % , at least about 55 % , at least lamidopropyl )dimethylammonio - 1 - propane sulfate about 60 % , at least about 65 % , at least about 70 % , specifi - (CHAPS ) , and 3 - ( 3 - chlolamidopropyl) dimethylammonio - 2 cally , a purity level of at least about 75 % , 80 % , 85 % , and 35 hydroxy - 1 - propane sulfonate (CHAPSO ) . Organic , water more specifically , a purity level of at least about 90 % , a miscible solvents such as acetonitrile , lower alkanols (espe purity level of at least about 95 % , a purity level of at least cially C2 -C4 alkanols such as ethanol or isopropanol) , or about 99 % or greater as determined by appropriate methods lower alkandiols ( especially C2 -C4 alkandiols such as eth such as SDS /PAGE analysis , RP -HPLC , SEC , and capillary ylene - glycol) may be used as denaturants. Phospholipids electrophoresis . 40 useful in the present invention may be naturally occurring A " recombinant host cell ” or " host cell” refers to a cell phospholipids such as phosphatidylethanolamine , phospha that includes an exogenous polynucleotide , regardless of the tidylcholine , phosphatidylserine , and phosphatidylinositol method used for insertion , for example , direct uptake , trans - or synthetic phospholipid derivatives or variants such as duction , f -mating , or other methods known in the art to dihexanoylphosphatidylcholine or diheptanoylphosphati create recombinant host cells . The exogenous polynucle - 45 dylcholine . otide may be maintained as a nonintegrated vector, for " Refolding , ” as used herein describes any process , reac example , a plasmid , or alternatively , may be integrated into tion or method which transforms disulfide bond containing the host genome. polypeptides from an improperly folded or unfolded state to As used herein , the term “ medium ” or “ media ” includes a native or properly folded conformation with respect to any culture medium , solution , solid , semi- solid , or rigid 50 disulfide bonds . support that may support or contain any host cell , including “ Cofolding, ” as used herein , refers specifically to refold bacterial host cells , yeast host cells , insect host cells , plant ing processes , reactions , or methods which employ at least host cells , eukaryotic host cells , mammalian host cells, CHO two polypeptides which interact with each other and result cells , prokaryotic host cells , E . coli , or Pseudomonas host in the transformation of unfolded or improperly folded cells , and cell contents . Thus, the term may encompass 55 polypeptides to native , properly folded polypeptides . medium in which the host cell has been grown, e . g . , medium The term “ proinsulin ” as used herein is a properly cross into which the relaxin polypeptide has been secreted , includ - line protein of the formula : ing medium either before or after a proliferation step . The term also may encompass buffers or reagents that contain B - C - A host cell lysates , such as in the case where the relaxin 60 wherein : polypeptide is produced intracellularly and the host cells are A is the A chain of relaxin or a functional derivative thereof; lysed or disrupted to release the relaxin polypeptide . B is the B chain of relaxin or a functional derivative thereof “ Reducing agent, ” as used herein with respect to protein having an . epsilon . - amino group ; and refolding, is defined as any compound or material which C is the connecting peptide of proinsulin . Preferably , pro maintains sulfhydryl groups in the reduced state and reduces 65 insulin is the A chain of human relaxin , the B chain ofhuman intra - or intermolecular disulfide bonds . Suitable reducing relaxin , and C is the natural connecting peptide . When agents include , but are not limited to , dithiothreitol (DTT ) , proinsulin is the natural sequence , proinsulin possesses three US 9 ,962 ,450 B2 23 24 free amino groups: Phenylalanine ( 1) (. alpha . -amino group ), wherein Pro at position B28 is substituted with Asp , Lys, Lysine (29 ) ( . epsilon . - amino group ) and Lysine( 64 ) (. epsi - Leu , Val , or Ala , and wherein Lys at position B29 is Lys or lon . - amino group ) . is substituted with Pro . Another monomeric relaxin analog , The term “ relaxin analog ” as used herein is a properly also known as des (B27 ) human relaxin , is human relaxin cross - lined protein exhibiting relaxin activity of the formula : 5 wherein Thr at position 27 of the B - chain is deleted . Monomeric relaxin analogs are disclosed in Chance , R . E . , A - B et al. , U . S . Pat. No . 5 , 514 ,646 , issued May 7 , 1996 ; Brems, wherein : D . N ., et al. Protein Engineering , 5, 527 -533 (1992 ); Brange, A is the A chain of relaxin or a functional derivative of the J . J . V . , et al ., EPO publication No. 214 ,826 ( published Mar . relaxin A chain ; and 10 18 , 1987 ) ; and Brange , J . J . V ., et al ., Current Opinion in B is the B chain of relaxin or a functional derivative of the Structural Biology, 1 , 934 - 940 ( 1991 ) . The monomeric relaxin B chain having an . epsilon . - amino group and at least relaxin analogs employed in the present formulations are one of A or B contains an amino acid modification from the properly cross - linked at the same positions as in human natural sequence . relaxin . In the present specification , whenever the term relaxin is 15 Relaxin peptides including , but not limited to , relaxin , used in a plural or a generic sense it is intended to encompass human ; relaxin , porcine ; IGF- I, human ; relaxin - like growth both naturally occurring insulins and relaxin analogues and factor II (69 - 84 ); pro -relaxin - like growth factor II (68 - 102 ), derivatives thereof. By " relaxin polypeptide” as used herein human ; pro - relaxin -like growth factor II ( 105 - 128 ), human ; is meant a compound having a molecular structure similar to [ AspB28 ] - relaxin , human ; [ LysB28 ] - relaxin , human ; that of human relaxin including the disulfide bridges 20 [LeuB28 ) -relaxin , human ; ValB28 ) -relaxin , human ; between Cys . sup .A7 and Cys . sup .B7 and between [AlaB28 ] - relaxin , human ; [AspB28 , ProB29 ] - relaxin , Cys. sup . A20 and Cys .sup . B19 and an internal disulfide human ; [LysB28 , ProB29 ] -relaxin , human ; [LeuB28 , bridge between Cys . sup .A6 and Cys. sup .A11 , and which ProB29 ] -relaxin , human ; [ ValB28 , ProB29 ] - relaxin , human ; have relaxin activity. [ AlaB28 , ProB29 ]- relaxin , human ; [GlyA21 ] -relaxin , The term " relaxin ” as used herein , refers to human 25 human ; [GlyA21 GlnB3] - relaxin , human ; [ AlaA21 ] - relaxin , relaxin , whose amino acid sequence and spatial structure are human ; [ AlaA21 Gln .sup .B3 ] relaxin , human ; [GlnB3 ] - re well - known . Human relaxin is comprised of a twenty - one laxin , human ; [GinB30 ] - relaxin , human ; [GlyA21 GluB301 amino acid A -chain and a thirty amino acid B -chain which relaxin , human ; (GlyA21 GinB3 GluB30 ) -relaxin , human ; are cross - linked by disulfide bonds. A properly cross - linked [GlnB3 GluB30 ) - relaxin , human ; B22 -B30 relaxin , human ; relaxin contains three disulfide bridges : one between posi - 30 B23 -B30 relaxin , human ; B25 -B30 relaxin , human ; B26 tion 7 of the A -chain and position 7 of the B -chain , a second B30 relaxin , human ; B27 -B30 relaxin , human ; B29 -B30 between position 20 of the A - chain and position 19 of the relaxin , human ; the A chain of human relaxin , and the B B - chain , and a third between positions 6 and 11 of the chain of human relaxin . A - chain [Nicol , D . S . H . W . and Smith , L . F . , Nature , 187 , In a further aspect , the invention provides recombinant 483 -485 (1960 ) ]. 35 nucleic acids encoding the variant proteins , expression vec Relaxin peptides including , but not limited to , relaxin , tors containing the variant nucleic acids, host cells compris human ; relaxin , porcine ; IGF- I , human ; relaxin - like growth ing the variant nucleic acids and /or expression vectors , and factor II (69 - 84 ) ; pro -relaxin - like growth factor II (68 - 102 ) , methods for producing the variant proteins. In an additional human ; pro - relaxin - like growth factor II ( 105 - 128 ) , human ; aspect, the invention provides treating a relaxin responsive [ AspB28 ] - relaxin , human ; [LysB28 ] - relaxin , human ; 40 disorder by administering to a patient a variant protein , [ LeuB28 ) -relaxin , human ; [ ValB28 ) -relaxin , human ; usually with a pharmaceutical carrier , in a therapeutically [ AlaB28 ] -relaxin , human ; [AspB28 , ProB29 ] -relaxin , effective amount. In a further aspect , the invention provides human ; [LysB28 , ProB29 ]- relaxin , human ; [LeuB28 , methods for modulating immunogenicity (particularly ProB29 ] - relaxin , human ; [ ValB28 , ProB29 ] - relaxin , human ; reducing immunogenicity ) of relaxin polypeptides by alter [ AlaB28 , ProB29 ]- relaxin , human ; [GlyA21 ]- relaxin , 45 ing MHC Class II epitopes. human ; [GlyA21 GinB3] - relaxin , human ; [ AlaA21 ] -relaxin , The term “ relaxin polypeptide ” also includes the pharma human ; [AlaA21 Gln .sup . B3 ] relaxin , human ; [GlnB3 ] - re - ceutically acceptable salts and prodrugs , and prodrugs of the laxin , human ; [GlnB30 ] - relaxin , human ; [GlyA21 GluB30 ] - salts , polymorphs , hydrates, solvates, biologically -active relaxin , human ; [GlyA21 GinB3 GluB30 ]- relaxin , human ; fragments , biologically active variants and stereoisomers of (GinB3 GluB301- relaxin , human ; B22 -B30 relaxin , human ; 50 the naturally - occurring relaxin as well as agonist, mimetic , B23 -B30 relaxin , human ; B25 - B30 relaxin , human ; B26 - and antagonist variants of the naturally - occurring relaxin B30 relaxin , human ; B27 - B30 relaxin , human ; B29 - B30 and polypeptide fusions thereof . Fusions comprising addi relaxin , human ; the A chain of human relaxin , and theB t ional amino acids at the amino terminus , carboxyl terminus , chain of human relaxin . or both , are encompassed by the term “ relaxin polypeptide .” The term " relaxin analog ” means a protein that has an 55 Exemplary fusions include, but are not limited to , e . g ., A - chain and a B - chain that have substantially the same methionyl relaxin in which a methionine is linked to the amino acid sequences as the A - chain and / or B - chain of N - terminus of relaxin resulting from the recombinant human relaxin , respectively , but differ from the A - chain and expression of the mature form of relaxin lacking the leader B - chain of human relaxin by having one or more amino acid or signal peptide or portion thereof ( a methionine is linked deletions , one or more amino acid replacements , and / or one 60 to the N - terminus of relaxin resulting from the recombinant or more amino acid additions that do not destroy the relaxin expression ), fusions for the purpose of purification ( includ activity of the relaxin analog . A relaxin analog having an ing, but not limited to , to poly - histidine or affinity epitopes) , isoelectric point that is “ higher than ” the isoelectric point of fusions with serum albumin binding peptides and fusions relaxin is one type of relaxin analog . Another type of relaxin with serum proteins such as serum albumin . U . S . Pat. No . analog is a " monomeric relaxin analog . " 65 5 ,750 ,373 , which is incorporated by reference herein , A " monomeric relaxin analog ” is a fast - acting analog of describes a method for selecting novel proteins such as human relaxin , including, for example , human relaxin growth hormone and antibody fragment variants having US 9 , 962, 450 B2 25 26 altered binding properties for their respective receptor mol encompassed by the term “ relaxin polypeptide. ” In some ecules . The method comprises fusing a gene encoding a embodiments , the relaxin antagonist comprises a non -natu protein of interest to the carboxy terminal domain of the rally encoded amino acid linked to a water soluble polymer gene III coat protein of the filamentous phage M13 . Chi that is present in a receptor binding region of the relaxin meric molecules comprising relaxin and one or more other 5 molecule . molecules . The chimeric molecule can contain specific In some embodiments , the relaxin polypeptides further regions or fragments of one or both of the relaxin and the comprise an addition , substitution or deletion thatmodulates other molecule( s ) . Any such fragments can be prepared from biological activity of the relaxin polypeptide. In some the proteins by standard biochemical methods , or by embodiments , the relaxin polypeptides further comprise an expressing a polynucleotide encoding the fragment. Relaxin , 10 addition , substitution or deletion that modulates anti - viral or a fragment thereof, can be produced as a fusion protein activity of the relaxin polypeptide . In some embodiments , comprising human serum albumin (HSA ) , Fc, or a portion the relaxin polypeptides further comprise an addition , sub thereof. Such fusion constructs are suitable for enhancing stitution or deletion that enhances anti - viral activity of the expression of the relaxin , or fragment thereof, in an eukary - relaxin polypeptide . For example , the additions , substitu otic host cell. Exemplary HSA portions include the N - ter - 15 tions or deletions may modulate one or more properties or minal polypeptide (amino acids 1 - 369, 1 -419 , and interme- activities of relaxin . For example , the additions, substitu diate lengths starting with amino acid 1 ) , as disclosed in U . S . tions or deletions may modulate affinity for the relaxin Pat . No. 5 , 766 ,883 , and publication WO 97 /24445 , which receptor, modulate circulating half - life , modulate therapeu are incorporated by reference herein . Other chimeric poly - tic half - life , modulate stability of the polypeptide , modulate peptides can include a HSA protein with relaxin , or frag - 20 cleavage by proteases, modulate dose , modulate release or ments thereof, attached to each of the C -terminal and bio -availability , facilitate purification , or improve or alter a N - terminal ends of the HSA . Such HSA constructs are particular route of administration . Similarly , relaxin poly disclosed in U . S . Pat. No . 5 ,876 , 969 , which is incorporated peptides may comprise protease cleavage sequences , reac by reference herein . Other fusions may be created by fusion tive groups , antibody - binding domains ( including but not of relaxin with a ) the Fc portion of an immunoglobulin ; b ) 25 limited to , FLAG or poly -His ) or other affinity based an analog of the Fc portion of an immunoglobulin ; and c ) sequences (including but not limited to , FLAG , poly -His , fragments of the Fc portion of an immunoglobulin . GST , etc . ) or linked molecules ( including but not limited to , Various references disclose modification of polypeptides biotin ) that improve detection (including but not limited to , by polymer conjugation or glycosylation . The term " relaxin GFP ) , purification or other traits of the polypeptide . polypeptide” includes polypeptides conjugated to a polymer 30 The term “ relaxin polypeptide ” also encompasses such as PEG and may be comprised of one or more addi- homodimers , heterodimers , homomultimers , and heteromul tional derivitizations of cysteine , lysine , or other residues . In timers that are linked , including but not limited to those addition , the relaxin polypeptide may comprise a linker or linked directly via non - naturally encoded amino acid side polymer , wherein the amino acid to which the linker or chains , either to the same or different non - naturally encoded polymer is conjugated may be a non - natural amino acid 35 amino acid side chains , to naturally - encoded amino acid side according to the present invention , or may be conjugated to chains , or indirectly via a linker. Exemplary linkers includ a naturally encoded amino acid utilizing techniques known ing but are not limited to , small organic compounds , water in the art such as coupling to lysine or cysteine . soluble polymers of a variety of lengths such as poly The term “ relaxin polypeptide” also includes glycosylated ( ethylene glycol) or polydextran , or polypeptides of various relaxin , such as but not limited to , polypeptides glycosylated 40 lengths . at any amino acid position , N - linked or O - linked glycosy A non -naturally encoded amino acid ” refers to an amino lated forms of the polypeptide . Variants containing single acid that is not one of the 20 common amino acids or nucleotide changes are also considered as biologically active pyrrolysine or selenocysteine . Other terms that may be used variants of relaxin polypeptide . In addition , splice variants synonymously with the term “ non -naturally encoded amino are also included . The term " relaxin polypeptide” also 45 acid ” are " non - natural amino acid ," " unnatural amino acid , " includes relaxin polypeptide heterodimers , homodimers, " non -naturally - occurring amino acid , " and variously heteromultimers , or homomultimers of any one or more hyphenated and non -hyphenated versions thereof. The term relaxin polypeptides or any other polypeptide , protein , car - " non - naturally encoded amino acid ” also includes , but is not bohydrate , polymer , small molecule , linker, ligand , or other limited to , amino acids that occur by modification ( e . g . biologically active molecule of any type , linked by chemical 50 post - translational modifications ) of a naturally encoded means or expressed as a fusion protein , as well as polypep - amino acid ( including but not limited to , the 20 common tide analogues containing, for example , specific deletions or amino acids or pyrrolysine and selenocysteine ) but are not other modifications yet maintain biological activity . themselves naturally incorporated into a growing polypep The term “ relaxin polypeptide ” or “ relaxin ” encompasses tide chain by the translation complex . Examples of such relaxin polypeptides comprising one or more amino acid 55 non - naturally -occurring amino acids include , but are not substitutions, additions or deletions. Relaxin polypeptides of limited to , N -acetylglucosaminyl - L - serine , N -acetylglu the present invention may be comprised of modifications cosaminyl- L - threonine , and O -phosphotyrosine . with one or more natural amino acids in conjunction with An “ amino terminus modification group ” refers to any one or more non - natural amino acid modification . Exem - molecule that can be attached to the amino terminus of a plary substitutions in a wide variety of amino acid positions 60 polypeptide . Similarly , a " carboxy terminus modification in naturally -occurring relaxin polypeptides have been group ” refers to any molecule that can be attached to the described , including but not limited to substitutions that carboxy terminus of a polypeptide . Terminus modification modulate pharmaceutical stability , that modulate one or groups include, but are not limited to , various water soluble more of the biological activities of the relaxin polypeptide , polymers , peptides or proteins such as serum albumin , or such as but not limited to, increase agonist activity, increase 65 other moieties that increase serum half - life of peptides . solubility of the polypeptide , decrease protease susceptibil - The terms “ functional group " , " active moiety ” , " activat ity , convert the polypeptide into an antagonist, etc . and are ing group ” , “ leaving group " , " reactive site ” , " chemically US 9 , 962 ,450 B2 27 28 reactive group " and " chemically reactive moiety ” are used agents , anti - inflammatory agents , anti -tumor agents , cardio in the art and herein to refer to distinct , definable portions or vascular agents , anti -anxiety agents , hormones, growth fac units of a molecule . The terms are somewhat synonymous in tors , steroidal agents , microbially derived toxins , and the the chemical arts and are used herein to indicate the portions like . of molecules that perform some function or activity and are 5 A " bifunctional polymer ” refers to a polymer comprising reactive with other molecules . two discrete functional groups that are capable of reacting The term " linkage " or " linker ” is used herein to refer to specifically with other moieties ( including but not limited to , groups or bonds that normally are formed as the result of a amino acid side groups ) to form covalent or non -covalent chemical reaction and typically are covalent linkages . linkages . A bifunctional linker having one functional group Hydrolytically stable linkages means that the linkages are 10 reactive with a group on a particular biologically active substantially stable in water and do not react with water at component, and another group reactive with a group on a useful pH values , including but not limited to , under physi second biological component, may be used to form a con ological conditions for an extended period of time, perhaps jugate that includes the first biologically active component, even indefinitely . Hydrolytically unstable or degradable the bifunctional linker and the second biologically active linkages mean that the linkages are degradable in water or in 15 component. Many procedures and linker molecules for aqueous solutions , including for example , blood . Enzymati - attachment of various compounds to peptides are known . cally unstable or degradable linkages mean that the linkage See , e . g . , European Patent Application No. 188 ,256 ; U . S . can be degraded by one or more enzymes . As understood in Pat. Nos. 4 ,671 , 958 , 4 ,659 , 839 , 4 ,414 , 148 , 4 ,699 , 784 ; the art , PEG and related polymers may include degradable 4 ,680 , 338 ; and 4 ,569 , 789 which are incorporated by refer linkages in the polymer backbone or in the linker group 20 ence herein . A “ multi - functional polymer” refers to a poly between the polymer backbone and one or more of the mer comprising two or more discrete functional groups that terminal functional groups of the polymer molecule . For are capable of reacting specifically with other moieties example , ester linkages formed by the reaction of PEG (including but not limited to , amino acid side groups) to carboxylic acids or activated PEG carboxylic acids with form covalent or non -covalent linkages. A bi- functional alcohol groups on a biologically active agent generally 25 polymer or multi - functional polymer may be any desired hydrolyze under physiological conditions to release the length or molecular weight , and may be selected to provide agent. Other hydrolytically degradable linkages include , but a particular desired spacing or conformation between one or are not limited to , carbonate linkages; imine linkages more molecules linked to the relaxin and its receptor or resulted from reaction of an amine and an aldehyde ; phos - relaxin . phate ester linkages formed by reacting an alcohol with a 30 Where substituent groups are specified by their conven phosphate group ; hydrazone linkages which are reaction tional chemical formulas, written from left to right, they product of a hydrazide and an aldehyde; acetal linkages that equally encompass the chemically identical substituents that are the reaction product of an aldehyde and an alcohol; would result from writing the structure from right to left , for orthoester linkages that are the reaction product of a formate example , the structure CH2O is equivalent to the structure and an alcohol; peptide linkages formed by an amine group , 35 OCH2 . including but not limited to , at an end of a polymer such as The term “ substituents ” includes but is not limited to PEG , and a carboxyl group of a peptide ; and oligonucleotide " non -interfering substituents ” . “Non - interfering substitu linkages formed by a phosphoramidite group , including but ents ” are those groups that yield stable compounds. Suitable not limited to , at the end of a polymer , and a 5 ' hydroxyl non - interfering substituents or radicals include, but are not group of an oligonucleotide . 40 limited to , halo , C1 -C10 alkyl , C2 -C10 alkenyl, C2 -C10 The term “ biologically active molecule ” , “ biologically alkynyl, C1 -C10 alkoxy , C1- C12 aralkyl, C1 -C12 alkaryl, active moiety ” or “ biologically active agent" when used C3 - C12 cycloalkyl, C3- C12 cycloalkenyl, phenyl, substi herein means any substance which can affect any physical or tuted phenyl, toluoyl, xylenyl, biphenyl, C2 -C12 alkoxy biochemical properties of a biological system , pathway, alkyl, C2 - C12 alkoxyaryl, C7- C12 aryloxyalkyl, C7 - C12 molecule, or interaction relating to an organism , including 45 oxyaryl, C1 - C6 alkylsulfinyl, C1- C10 alkylsulfonyl , but not limited to , viruses, bacteria , bacteriophage , transpo (CH2 ) m - 0 (C1 -C10 alkyl) wherein m is from 1 to 8 , son , prion , insects , fungi, plants , animals , and humans. In aryl, substituted aryl, substituted alkoxy , fluoroalkyl , het particular, as used herein , biologically active molecules erocyclic radical , substituted heterocyclic radical, nitroalkyl, include , but are not limited to , any substance intended for — NO2, CN , — NRC ( O ) - (C1 -C10 alkyl) , - C ( O ) diagnosis , cure , mitigation , treatment, or prevention of dis - 50 (C1 -C10 alkyl ) , C2- C10 alkyl thioalkyl, - C ( O ) O - (C1 ease in humans or other animals , or to otherwise enhance C10 alkyl) , OH , SO2 , = S , COOH , - NR2 , carbo physical or mental well -being of humans or animals . nyl , - C ( O ) - (C1 -C10 alkyl) -CF3 , C ( O ) - CF3, C (O ) Examples of biologically active molecules include, but are NR2, (C1 -C10 aryl) - S (C6 -C10 aryl) , C ( O ) - (C1 not limited to , peptides , proteins, enzymes, small molecule C10 aryl) , (CH2 )m 0 - 4 (CH2 ) m - 0 (C1 - C10 alkyl) drugs, vaccines, immunogens, hard drugs, soft drugs, car- 55 wherein each m is from 1 to 8 , - C ( O )NR2 , C (S )NR2 , bohydrates, inorganic atoms or molecules, dyes, lipids, SO2NR2, - NRC ( O ) NR2 , — NRC ( S ) NR2, salts thereof, nucleosides , radionuclides, oligonucleotides , toxoids, tox - and the like . Each R as used herein is H , alkyl or substituted ins, prokaryotic and eukaryotic cells , viruses, polysaccha alkyl, aryl or substituted aryl , aralkyl , or alkaryl. rides, nucleic acids and portions thereof obtained or derived The term “ halogen ” includes fluorine , chlorine , iodine , from viruses , bacteria , insects , animals or any other cell or 60 and bromine . cell type , liposomes, microparticles and micelles . The T he term “ alkyl, ” by itself or as part of another substitu relaxin polypeptides may be added in a micellular formu - ent, means, unless otherwise stated , a straight or branched lation ; see U .S . Pat . No . 5 ,833 ,948 , which is incorporated by chain , or cyclic hydrocarbon radical, or combination thereof, reference herein in its entirety . Classes ofbiologically active which may be fully saturated , mono - or polyunsaturated and agents that are suitable for use with the invention include, 65 can include di- and multivalent radicals , having the number but are not limited to , drugs , prodrugs , radionuclides, imag - of carbon atoms designated ( i . e . C1 -C10 means one to ten ing agents , polymers , antibiotics , fungicides , anti- viral carbons) . Examples of saturated hydrocarbon radicals US 9 ,962 ,450 B2 29 30 include, but are not limited to , groups such as methyl, ethyl, cloalkyl, a heteroatom can occupy the position at which the n -propyl , isopropyl, n - butyl, t - butyl, isobutyl, sec -butyl , heterocycle is attached to the remainder of the molecule . cyclohexyl, (cyclohexyl ) methyl , cyclopropylmethyl, Examples of cycloalkyl include , but are not limited to , homologs and isomers of, for example, n -pentyl , n - hexyl, cyclopentyl, cyclohexyl , 1 -cyclohexenyl , 3 - cyclohexenyl, n -heptyl , n -octyl , and the like . An unsaturated alkyl group is 5 cycloheptyl, and the like . Examples of heterocycloalkyl one having one or more double bonds or triple bonds. include , but are not limited to , 1 - ( 1 , 2 , 5 , 6 - tetrahydropyridyl) , Examples of unsaturated alkyl groups include , but are not 1 -piperidinyl , 2 - piperidinyl, 3 - piperidinyl , 4 -morpholinyl , limited to , vinyl, 2 - propenyl, crotyl, 2 - isopentenyl, 2 - (buta 3 -morpholinyl , tetrahydrofuran - 2 -yl , tetrahydrofuran - 3 - yl, dienyl ) , 2 , 4 -pentadienyl , 3 - ( 1 , 4 -pentadienyl ) , ethynyl, 1 - tetrahydrothien - 2 - yl, tetrahydrothien - 3 - yl, 1 -piperazinyl , and 3 - propynyl, 3 -butynyl , and the higher homologs and 10 2 -piperazinyl , and the like . Additionally , the term encom isomers . The term “ alkyl, ” unless otherwise noted , is also passes bicyclic and tricyclic ring structures . Similarly , the meant to include those derivatives of alkyl defined in more term " heterocycloalkylene ” by itself or as part of another detail below , such as “ heteroalkyl. ” Alkyl groups which are substituent means a divalent radical derived from heterocy limited to hydrocarbon groups are termed “ homoalkyl” . cloalkyl, and the term “ cycloalkylene ” by itself or as part of The term “ alkylene” by itself or as part of another 15 another substituent means a divalent radical derived from substituent means a divalent radical derived from an alkane , cycloalkyl. as exemplified , but not limited , by the structures As used herein , the term " water soluble polymer ” refers _ CH2CH2 - and - CH2CH2CH2CH2 -, and further to any polymer that is soluble in aqueous solvents . Linkage includes those groups described below as " heteroalkylene ." of water soluble polymers to relaxin polypeptides can result Typically , an alkyl ( or alkylene ) group will have from 1 to 20 in changes including, but not limited to , increased ormodu 24 carbon atoms, with those groups having 10 or fewer lated serum half - life , or increased or modulated therapeutic carbon atoms being a particular embodiment of the methods half- life relative to the unmodified form , modulated immu and compositions described herein . A “ lower alkyl” or nogenicity , modulated physical association characteristics “ lower alkylene ” is a shorter chain alkyl or alkylene group , such as aggregation and multimer formation , altered recep generally having eight or fewer carbon atoms. 25 tor binding, altered binding to one or more binding partners , The terms “ alkoxy , ” “ alkylamino ” and “ alkylthio ” ( or and altered receptor dimerization or multimerization . The thioalkoxy ) are used in their conventional sense , and refer to water soluble polymer may or may not have its own bio those alkyl groups attached to the remainder of the molecule logical activity , and may be utilized as a linker for attaching via an oxygen atom , an amino group , or a sulfur atom , relaxin to other substances, including but not limited to one respectively . 30 or more relaxin polypeptides, or one or more biologically The term " heteroalkyl, ” by itself or in combination with active molecules. Suitable polymers include , but are not another term , means, unless otherwise stated , a stable limited to , polyethylene glycol, polyethylene glycol propi straight or branched chain , or cyclic hydrocarbon radical, or o naldehyde , mono C1 - C10 alkoxy or aryloxy derivatives combinations thereof, consisting of the stated number of thereof (described in U . S . Pat. No . 5, 252 ,714 which is carbon atoms and at least one heteroatom selected from the 35 incorporated by reference herein ) , monomethoxy -polyeth group consisting of O , N , Si and S , and wherein the nitrogen ylene glycol, polyvinyl pyrrolidone , polyvinyl alcohol, and sulfur atoms may optionally be oxidized and the nitro - polyamino acids, divinylether maleic anhydride, N - (2 -Hy gen heteroatom may optionally be quaternized . The heteroa - droxypropyl) -methacrylamide , dextran , dextran derivatives tom ( s ) O , N and S and Si may be placed at any interior including dextran sulfate , polypropylene glycol, polypropyl position of the heteroalkyl group or at the position at which 40 ene oxide/ ethylene oxide copolymer , polyoxyethylated the alkyl group is attached to the remainder of the molecule . polyol, heparin , heparin fragments, polysaccharides , oli Examples include , but are not limited to , - CH2 -CH2 - 0 gosaccharides, glycans , cellulose and cellulose derivatives , CH3, CH2 - CH2- NHCH3, CH2 - CH2- N ( CH3 )- CH3 , including but not limited to methylcellulose and carboxym - CH2 - S - CH2 -CH3 , CH2 -CH2 , - S ( O ) - CH3, ethyl cellulose, starch and starch derivatives , polypeptides , - CH2 -CH2 - S ( O ) 2 -CH3 , _ CH _ CHO CH3 , Si 45 polyalkylene glycol and derivatives thereof, copolymers of ( CH3 ) 3 , CH2- CH = N - OCH3, and CH = CH - N polyalkylene glycols and derivatives thereof, polyvinyl ethyl (CH3 ) -CH3 . Up to two heteroatoms may be consecutive , ethers , and alpha -beta - poly [ ( 2 - hydroxyethyl ) -DL - aspart such as, for example , - CH2- NH - OCH3 and - CH2- 0 amide, and the like, or mixtures thereof. Examples of such Si( CH3 )3 . Similarly , the term " heteroalkylene ” by itself or water soluble polymers include , but are not limited to , as part of another substituent means a divalent radical 50 polyethylene glycol and serum albumin . derived from heteroalkyl, as exemplified , but not limited by, As used herein , the term “ polyalkylene glycol” or “ poly - CH2 - CH2 -S _ CH2 CH2 - and CH2 -S _ CH2 -CH2 - (alkene glycol) ” refers to polyethylene glycol (poly ( ethylene NH - CH2 - . For heteroalkylene groups , the same or differ- glycol) ) , polypropylene glycol, polybutylene glycol, and ent heteroatoms can also occupy either or both of the chain derivatives thereof. The term “ polyalkylene glycol” encom termini ( including but not limited to , alkyleneoxy, alkylene - 55 passes both linear and branched polymers and average dioxy, alkyleneamino , alkylenediamino , aminooxyalkylene , molecular weights of between 0 . 1 kDa and 100 kDa. Other and the like ) . Still further , for alkylene and heteroalkylene exemplary embodiments are listed , for example, in commer linking groups, no orientation of the linking group is implied cial supplier catalogs , such as Shearwater Corporation 's by the direction in which the formula of the linking group is catalog “ Polyethylene Glycol and Derivatives for Biomedi written . For example , the formula - C ( O )2R ' represents 60 cal Applications” ( 2001) . both - C ( O ) 2R ' and — R ' C ( O ) 2 . The term “ aryl” means , unless otherwise stated , a poly The terms “ cycloalkyl” and “ heterocycloalkyl” , by them unsaturated , aromatic , hydrocarbon substituent which can be selves or in combination with other terms, represent, unless a single ring or multiple rings ( including but not limited to , otherwise stated , cyclic versions of alkyl” and “ het - from 1 to 3 rings ) which are fused together or linked eroalkyl” , respectively . Thus, a cycloalkyl or heterocy - 65 covalently . The term “ heteroaryl” refers to aryl groups (or cloalkyl include saturated , partially unsaturated and fully rings ) that contain from one to four heteroatoms selected unsaturated ring linkages. Additionally , for heterocy from N , O , and S , wherein the nitrogen and sulfur atoms are US 9 , 962, 450 B2 31 32 optionally oxidized , and the nitrogen atom ( s ) are optionally = NR ', = N / OR ', - NR 'R " , - SR ', -halogen , - SiR ' R " R " , quaternized . A heteroaryl group can be attached to the OC ( O ) R ', - C ( O ) R ' , CO2R ', CONR' R " , - OC (O )NR 'R " , remainder of the molecule through a heteroatom . Non - NR " C (O )R ', NR 'C (O )NR " R "" , — NR " C (O ) 2R ', NR - C limiting examples of aryl and heteroaryl groups include (NR ' R " R '" ) = NR " " , NR C (NR ' R " ) — NR "" , - S ( O ) R ', phenyl, 1 -naphthyl , 2 - naphthyl, 4 -biphenyl , 1 -pyrrolyl , 5 - S ( O )2R ' , - S ( O ) 2NR 'R " . NRSO2R ', - CN and — NO2, 2 - pyrrolyl , 3 -pyrrolyl , 3 -pyrazolyl , 2 - imidazolyl, 4 - imida - R ', N3 , - CH ( Ph )2 , fluoro (C1 - C4 )alkoxy , and fluoro zolyl, pyrazinyl , 2 - oxazolyl , 4 - oxazolyl , 2 - phenyl- 4 - ox azolyl, 5 -oxazolyl , 3 - isoxazolyl, 4 - isoxazolyl, 5 - isoxazolyl, (C1 - C4 )alkyl , in a number ranging from zero to the total 2 - thiazolyl, 4 - thiazolyl, 5 -thiazolyl , 2 - furyl , 3 - furyl , 2 - thie number of open valences on the aromatic ring system ; and nyl, 3 -thienyl , 2 -pyridyl , 3 -pyridyl , 4 -pyridyl , 2 -pyrimidyl , 10 where R ', R " , R '' and R " " are independently selected from 4 -pyrimidyl , 5 -benzothiazolyl , purinyl, 2 - benzimidazolyl, hydrogen , alkyl, heteroalkyl, aryl and heteroaryl. When a 5 - indolyl , 1 - isoquinolyl, 5 - isoquinolyl, 2 - quinoxalinyl, compound of the invention includes more than one R group , 5 - quinoxalinyl, 3 - quinolyl, and 6 - quinolyl. Substituents for for example , each of the R groups is independently selected each of the above noted aryl and heteroaryl ring systems are as are each R ', R " , R '" and R " " groups when more than one selected from the group of acceptable substituents described 15 of these groups is present. below . As used herein , the term “ modulated serum half -life ” For brevity , the term “ aryl” when used in combination means the positive or negative change in circulating half - life with other terms ( including but not limited to , aryloxy, of a modified relaxin relative to its non -modified form . arylthioxy, arylalkyl) includes both aryl and heteroaryl rings Serum half - life is measured by taking blood samples at as defined above. Thus , the term “ arylalkyl” is meant to 20 various time points after administration of relaxin , and include those radicals in which an aryl group is attached to determining the concentration of that molecule in each an alkyl group (including but not limited to , benzyl, phen - sample . Correlation of the serum concentration with time ethyl, pyridylmethyl and the like ) including those alkyl allows calculation of the serum half- life. Increased serum groups in which a carbon atom ( including but not limited to , half- life desirably has at least about two -fold , but a smaller a methylene group ) has been replaced by, for example , an 25 increase may be useful, for example where it enables a oxygen atom ( including but not limited to , phenoxymethyl, satisfactory dosing regimen or avoids a toxic effect . In some 2 -pyridyloxymethyl , 3 - ( 1 -naphthyloxy ) propyl, and the like ) . embodiments , the increase is at least about three - fold , at Each of the above terms (including but not limited to , least about five - fold , or at least about ten - fold . “ alkyl, " " heteroalkyl, ” “ aryl” and “ heteroaryl” ) are meant to The term “ modulated therapeutic half - life ” as used herein include both substituted and unsubstituted forms of the 30 means the positive or negative change in the half - life of the indicated radical. Exemplary substituents for each type of therapeutically effective amount of relaxin , relative to its radical are provided below . non -modified form . Therapeutic half - life is measured by Substituents for the alkyl and heteroalkyl radicals ( includ measuring pharmacokinetic and / or pharmacodynamic prop ing those groups often referred to as alkylene , alkenyl, erties of the molecule at various time points after adminis heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, hetero - 35 tration . Increased therapeutic half - life desirably enables a cycloalkyl, cycloalkenyl, and heterocycloalkenyl ) can be particular beneficial dosing regimen , a particular beneficial one or more of a variety of groups selected from , but not total dose , or avoids an undesired effect. In some embodi limited to : — OR ', = 0 , = NR ', = N / OR ', - NR 'R " , - SR ', ments , the increased therapeutic half- life results from -halogen , — SiR ' R " R "" , OC ( O ) R ', - C ( O ) R ', CO2R ', increased potency , increased or decreased binding of the - CONR 'R ", OC (O )NR 'R ", — NR " C (O )R ', NR 'C (O ) 40 modified molecule to its target , increased or decreased NR " R '" , — NR " C (O )2R ', — NR - C (NR ' R " R " ) = NR '" , NR breakdown of the molecule by enzymes such as proteases , or C (NR ' R " ) — NR " , - S ( O ) R ', - S ( O ) 2R ', - S ( O ) 2NR ' R " , an increase or decrease in another parameter or mechanism NRSO2R ', - CN and — NO2 in a number ranging from zero of action of the non -modified molecule or an increase or to (2m ' + 1 ) , where m ' is the total number of carbon atoms in decrease in receptor -mediated clearance of the molecule . such a radical. R ', R " , R '' and R " " each independently refer45 The term “ isolated , " when applied to a nucleic acid or to hydrogen , substituted or unsubstituted heteroalkyl, sub - protein , denotes that the nucleic acid or protein is free of at stituted or unsubstituted aryl, including but not limited to , least some of the cellular components with which it is aryl substituted with 1 - 3 halogens , substituted or unsubsti - associated in the natural state , or that the nucleic acid or tuted alkyl , alkoxy or thioalkoxy groups, or arylalkyl protein has been concentrated to a level greater than the groups . When a compound of the invention includes more 50 concentration of its in vivo or in vitro production . It can be than one R group , for example , each of the R groups is in a homogeneous state . Isolated substances can be in either independently selected as are each R ', R " , R " " and R " " a dry or semi- dry state , or in solution , including but not groups when more than one of these groups is present . When limited to , an aqueous solution . It can be a component of a R ' and R " are attached to the same nitrogen atom , they can pharmaceutical composition that comprises additional phar be combined with the nitrogen atom to form a 5 -, 6 -, or 55 maceutically acceptable carriers and /or excipients . Purity 7 -membered ring . For example , — NR ' R " is meant to and homogeneity are typically determined using analytical include , but not be limited to , 1 -pyrrolidinyl and 4 -mor chemistry techniques such as polyacrylamide gel electro pholinyl. From the above discussion of substituents , one of phoresis or high performance liquid chromatography. A skill in the art will understand that the term “ alkyl” is meant protein which is the predominant species present in a to include groups including carbon atoms bound to groups 60 preparation is substantially purified . In particular, an isolated other than hydrogen groups , such as haloalkyl ( including but gene is separated from open reading frames which flank the not limited to , CF3 and - CH2CF3 ) and acyl ( including gene and encode a protein other than the gene of interest. but not limited to , C ( O )CH3 , - C ( O )CF3 , C ( O ) The term “ purified ” denotes that a nucleic acid or protein CH2OCH3, and the like ) . gives rise to substantially one band in an electrophoretic gel . Similar to the substituents described for the alkyl radical, 65 Particularly , it may mean that the nucleic acid or protein is substituents for the aryl and heteroaryl groups are varied and at least 85 % pure, at least 90 % pure , at least 95 % pure , at are selected from , but are not limited to : halogen , OR , = 0 , least 99 % or greater pure . US 9 , 962, 450 B2 33 34 The term “ nucleic acid ” refers to deoxyribonucleotides, of non -natural amino acids, including synthetic non -native deoxyribonucleosides , ribonucleosides, or ribonucleotides amino acids, substituted amino acids, or one or more and polymers thereof in either single - or double - stranded D -amino acids into the proteins of the present invention may form . Unless specifically limited , the term encompasses be advantageous in a number of different ways. D - amino nucleic acids containing known analogues of natural nucleo - 5 acid - containing peptides , etc . , exhibit increased stability in tides which have similar binding properties as the reference vitro or in vivo compared to L -amino acid - containing coun nucleic acid and are metabolized in a manner similar to terparts . Thus , the construction of peptides , etc ., incorporat naturally occurring nucleotides . Unless specifically limited ing D -amino acids can be particularly useful when greater otherwise , the term also refers to oligonucleotide analogs intracellular stability is desired or required . More specifi including PNA ( peptidonucleic acid ) , analogs of DNA used 10 cally, D - peptides, etc. , are resistant to endogenous pepti in antisense technology (phosphorothioates , phosphoroami- dases and proteases, thereby providing improved bioavail dates , and the like ) . Unless otherwise indicated , a particular ability of the molecule , and prolonged lifetimes in vivo nucleic acid sequence also implicitly encompasses conser - when such properties are desirable . Additionally , D -pep vatively modified variants thereof ( including but not limited tides , etc . , cannot be processed efficiently for major histo to , degenerate codon substitutions ) and complementary 15 compatibility complex class II -restricted presentation to T sequences as well as the sequence explicitly indicated . helper cells , and are therefore , less likely to induce humoral Specifically , degenerate codon substitutions may be immune responses in the whole organism . achieved by generating sequences in which the third position Amino acids may be referred to herein by either their of one or more selected (or all ) codons is substituted with commonly known three letter symbols or by the one -letter mixed -base and / or deoxyinosine residues (Batzer et al ., 20 symbols recommended by the IUPAC - IUB Biochemical Nucleic Acid Res. 19 : 5081 (1991 ) ; Ohtsuka et al ., J . Biol. Nomenclature Commission . Nucleotides , likewise , may be Chem . 260 : 2605 - 2608 ( 1985 ) ; Rossolini et al. , Mol. Cell . referred to by their commonly accepted single - letter codes . Probes 8 : 91 - 98 ( 1994 ) ) . " Conservatively modified variants " applies to both amino The terms " polypeptide ,” “ peptide ” and “ protein ” are acid and nucleic acid sequences . With respect to particular used interchangeably herein to refer to a polymer of amino 25 nucleic acid sequences , “ conservatively modified variants " acid residues. That is , a description directed to a polypeptide refers to those nucleic acids which encode identical or applies equally to a description of a peptide and a description essentially identical amino acid sequences, or where the of a protein , and vice versa . The terms apply to naturally nucleic acid does not encode an amino acid sequence , to occurring amino acid polymers as well as amino acid essentially identical sequences . Because of the degeneracy polymers in which one or more amino acid residues is a 30 of the genetic code , a large number of functionally identical non - naturally encoded amino acid . As used herein , the terms nucleic acids encode any given protein . For instance , the encompass amino acid chains of any length , including full codons GCA , GCC , GCG and GCU all encode the amino length proteins , wherein the amino acid residues are linked acid alanine. Thus , at every position where an alanine is by covalent peptide bonds. specified by a codon , the codon can be altered to any of the The term “ amino acid ” refers to naturally occurring and 35 corresponding codons described without altering the non -naturally occurring amino acids, as well as amino acid e ncoded polypeptide . Such nucleic acid variations are analogs and amino acid mimetics that function in a manner " silent variations, " which are one species of conservatively similar to the naturally occurring amino acids . Naturally modified variations. Every nucleic acid sequence herein encoded amino acids are the 20 common amino acids which encodes a polypeptide also describes every possible ( alanine , arginine, asparagine , aspartic acid , cysteine , glu - 40 silent variation of the nucleic acid . One of ordinary skill in tamine , glutamic acid , glycine, histidine, isoleucine , leucine , the art will recognize that each codon in a nucleic acid lysine, methionine, phenylalanine, proline , serine , threonine, ( except AUG , which is ordinarily the only codon for methio tryptophan , tyrosine , and valine) and pyrrolysine and sele - nine , and TGG , which is ordinarily the only codon for nocysteine . Amino acid analogs refers to compounds that tryptophan ) can be modified to yield a functionally identical have the same basic chemical structure as a naturally occur - 45 molecule . Accordingly , each silent variation of a nucleic ring amino acid , i. e ., an a carbon that is bound to a acid which encodes a polypeptide is implicit in each hydrogen , a carboxyl group , an amino group , and an R described sequence . group , such as, homoserine , norleucine , methionine sulfox - As to amino acid sequences , one of ordinary skill in the ide , methionine methyl sulfonium . Such analogs have modi- art will recognize that individual substitutions , deletions or fied R groups ( such as , norleucine ) or modified peptide 50 additions to a nucleic acid , peptide, polypeptide , or protein backbones , but retain the same basic chemical structure as a sequence which alters , adds or deletes a single amino acid or naturally occurring amino acid . Reference to an amino acid a small percentage of amino acids in the encoded sequence includes , for example , naturally occurring proteogenic is a " conservatively modified variant” where the alteration L - amino acids ; D - amino acids , chemically modified amino results in the deletion of an amino acid , addition of an amino acids such as amino acid variants and derivatives , naturally 55 acid , or substitution of an amino acid with a chemically occurring non - proteogenic amino acids such as B - alanine, similar amino acid . Conservative substitution tables provid ornithine, etc .; and chemically synthesized compounds hav - ing functionally similar amino acids are known to those of ing properties known in the art to be characteristic of amino ordinary skill in the art. Such conservatively modified vari acids. Examples of non - naturally occurring amino acids ants are in addition to and do not exclude polymorphic include , but are not limited to , a -methyl amino acids ( e . g ., 60 variants , interspecies homologs , and alleles of the invention . a -methyl alanine ) , D - amino acids, histidine - like amino Conservative substitution tables providing functionally acids (e . g. , 2 -amino -histidine , B -hydroxy -histidine , homo- similar amino acids are known to those of ordinary skill in histidine, a - fluoromethyl- histidine and a -methyl - histidine ), the art. The following eight groups each contain amino acids amino acids having an extra methylene in the side chain that are conservative substitutions for one another : (“ homo” amino acids ) , and amino acids in which a carbox - 65 1 ) Alanine ( A ) , Glycine ( G ) ; ylic acid functional group in the side chain is replaced with 2 ) Aspartic acid ( D ) , Glutamic acid ( E ) ; a sulfonic acid group ( e . g ., cysteic acid ). The incorporation 3 ) Asparagine (N ), Glutamine (Q ) ; US 9 , 962 , 450 B2 35 36 4 ) Arginine ( R ), Lysine ( K ) ; alignment and visual inspection ( see , e . g ., Ausubel et al. , 5 ) Isoleucine (I ) , Leucine ( L ) , Methionine ( M ), Valine Current Protocols in Molecular Biology ( 1995 supplement) ) . One example of an algorithm that is suitable for deter 6 ) Phenylalanine ( F ), Tyrosine ( Y ) , Tryptophan ( W ) ; mining percent sequence identity and sequence similarity 7 ) Serine ( S ), Threonine ( T ) ; and 5 are the BLAST and BLAST 2 . 0 algorithms, which are 8 ) Cysteine (C ), Methionine ( M ) described in Altschul et al. ( 1997) Nuc . Acids Res. 25 : 3389 3402 , and Altschul et al . ( 1990 ) J . Mol . Biol. 215 : 403 -410 , ( see, e . g . , Creighton , Proteins: Structures and Molecular respectively . Software for performing BLAST analyses is Properties ( W H Freeman & Co .; 2nd edition (December publicly available through the National Center for Biotech 1993 ). 10 nology Information available at the World Wide Web at The terms “ identical” or percent “ identity ,” in the context ncbi. nlm .nih . gov . The BLAST algorithm parameters W , T , of two or more nucleic acids or polypeptide sequences , refer and X determine the sensitivity and speed of the alignment. to two or more sequences or subsequences that are the same. The BLASTN program ( for nucleotide sequences ) uses as Sequences are " substantially identical” if they have a per defaults a wordlength ( W ) of 11 , an expectation ( E ) or 10 , centage of amino acid residues or nucleotides that are the 15 M = 5 . N = - 4 and a comparison of both strands. For amino same (i . e. , about 60 % identity, about 65 % , about 70 % , about acid sequences , the BLASTP program uses as defaults a 73 % , about 80 % , about 85 % , about 90 % , or about 95 % wordlength of 3 , and expectation ( E ) of 10 , and the BLO identity over a specified region ), when compared and SUM62 scoring matrix (see Henikoff and Henikoff ( 1992 ) aligned for maximum correspondence over a comparison Proc . Natl . Acad . Sci. USA 89 : 10915 ) alignments ( B ) of 50 , window , or designated region as measured using one of the 20 expectation ( E ) of 10 , M = 5, N = 4 , and a comparison of both following sequence comparison algorithms ( or other algo - strands. The BLAST algorithm is typically performed with rithms available to persons of ordinary skill in the art ) or by the “ low complexity ” filter turned off . manual alignment and visual inspection . This definition also The BLAST algorithm also performs a statistical analysis refers to the complement of a test sequence . The identity can of the similarity between two sequences (see , e . g . , Karlin exist over a region that is at least about 50 amino acids or 25 and Altschul (1993 ) Proc . Natl. Acad . Sci. USA 90 :5873 nucleotides in length , or over a region that is 75 - 100 amino 5787 ) . One measure of similarity provided by the BLAST acids or nucleotides in length , or, where not specified , across algorithm is the smallest sum probability (P ( N ) ) , which the entire sequence of a polynucleotide or polypeptide . A provides an indication of the probability by which a match polynucleotide encoding a polypeptide of the present inven - between two nucleotide or amino acid sequences would tion , including homologs from species other than human , 30 occur by chance . For example , a nucleic acid is considered may be obtained by a process comprising the steps of similar to a reference sequence if the smallest sum prob screening a library under stringent hybridization conditions ability in a comparison of the test nucleic acid to the with a labeled probe having a polynucleotide sequence of reference nucleic acid is less than about 0 . 2 , or less than the invention or a fragment thereof, and isolating full - length about 0 . 01, or less than about 0 . 001. cDNA and genomic clones containing said polynucleotide 35 The phrase " selectively (or specifically ) hybridizes to ” sequence. Such hybridization techniques are well known to refers to the binding , duplexing, or hybridizing of a mol the skilled artisan . ecule only to a particular nucleotide sequence under strin For sequence comparison , typically one sequence acts as gent hybridization conditions when that sequence is present a reference sequence, to which test sequences are compared in a complex mixture ( including but not limited to , total When using a sequence comparison algorithm , test and 40 cellular or library DNA or RNA ) . reference sequences are entered into a computer, subse The phrase " stringent hybridization conditions ” refers to quence coordinates are designated , if necessary , and hybridization of sequences of DNA , RNA, PNA , or other sequence algorithm program parameters are designated . nucleic acid mimics , or combinations thereof under condi Default program parameters can be used , or alternative tions of low ionic strength and high temperature as is known parameters can be designated . The sequence comparison 45 in the art . Typically , under stringent conditions a probe will algorithm then calculates the percent sequence identities for hybridize to its target subsequence in a complex mixture of the test sequences relative to the reference sequence, based nucleic acid ( including but not limited to , total cellular or on the program parameters . library DNA or RNA ) but does not hybridize to other A “ comparison window ” , as used herein , includes refer - sequences in the complex mixture . Stringent conditions are ence to a segment of any one of the number of contiguous 50 sequence - dependent and will be different in different cir positions selected from the group consisting of from 20 to cumstances . Longer sequences hybridize specifically at 600 , usually about 50 to about 200 , more usually about 100 higher temperatures . An extensive guide to the hybridization to about 150 in which a sequence may be compared to a of nucleic acids is found in Tijssen , Laboratory Techniques reference sequence of the same number of contiguous posi - in Biochemistry and Molecular Biology -Hybridization with tions after the two sequences are optimally aligned . Methods 55 Nucleic Probes , " Overview of principles of hybridization of alignment of sequences for comparison are known to and the strategy of nucleic acid assays ” ( 1993 ) . Generally , those of ordinary skill in the art . Optimal alignment of stringent conditions are selected to be about 5 - 10° C . lower sequences for comparison can be conducted , including but than the thermal melting point ( Tm ) for the specific not limited to , by the local homology algorithm of Smith and sequence at a defined ionic strength pH . The Tm is the Waterman ( 1970 ) Adv. Appl. Math . 2 : 482c , by the homol- 60 temperature (under defined ionic strength , pH , and nucleic ogy alignment algorithm of Needleman and Wunsch ( 1970 ) concentration ) at which 50 % of the probes complementary J . Mol. Biol. 48 :443 , by the search for similarity method of to the target hybridize to the target sequence at equilibrium Pearson and Lipman (1988 ) Proc . Nat’ l. Acad . Sci. USA (as the target sequences are present in excess, at Tm , 50 % of 85 : 2444 , by computerized implementations of these algo - the probes are occupied at equilibrium ) . Stringent conditions rithms (GAP , BESTFIT, FASTA , and TFASTA in the Wis - 65 may be those in which the salt concentration is less than consin Genetics Software Package , Genetics Computer about 1 . 0 M sodium ion , typically about 0 . 01 to 1 . 0 M Group , 575 Science Dr. , Madison , Wis .) , or by manual sodium ion concentration (or other salts ) at pH 7 .0 to 8 .3 and US 9 , 962 , 450 B2 37 38 the temperature is at least about 30° C . for short probes The term " post - translationally modified ” refers to any ( including but not limited to , 10 to 50 nucleotides ) and at modification of a natural or non -natural amino acid that least about 60° C . for long probes ( including but not limited occurs to such an amino acid after it has been incorporated to , greater than 50 nucleotides ) . Stringent conditions may into a polypeptide chain . The term encompasses , by way of also be achieved with the addition of destabilizing agents 5 example only , co -translational in vivo modifications , co such as formamide. For selective or specific hybridization , a translational in vitro modifications (such as in a cell- free positive signal may be at least two times background , translation system ) , post- translational in vivo modifications , and post - translational in vitro modifications. optionally 10 times background hybridization . Exemplary In prophylactic applications, compositions containing the stringent hybridization conditions can be as following : 50 % relaxin polypeptide are administered to a patient susceptible formamide , 5xSSC , and 1 % SDS , incubating at 42° C ., or 10 to or otherwise at risk of a particular disease , disorder or 5xSSC , 1 % SDS , incubating at 65° C . , with wash in condition . Such an amount is defined to be a " prophylacti 0 .2xSSC , and 0 . 1 % SDS at 65° C . Such washes can be cally effective amount .” In this use , the precise amounts also performed for 5 , 15 , 30 , 60 , 120 , or more minutes . depend on the patient' s state of health , weight, and the like . As used herein , the term " eukaryote ” refers to organisms. It is considered well within the skill of the art for one to belonging to the phylogenetic domain Eucarya such as 15 determine such prophylactically effective amounts by rou animals ( including but not limited to , mammals , insects , tine experimentation ( e . g ., a dose escalation clinical trial) . reptiles, birds, etc .) , ciliates, plants ( including but not lim The term “ protected ” refers to the presence of a “ protect ited to , monocots , dicots, algae, etc .) , fungi, yeasts , flagel- ing group ” or moiety that prevents reaction of the chemi lates , microsporidia , protists , etc . cally reactive functional group under certain reaction con As used herein , the term " non -eukaryote ” refers to non - 20 ditions . The protecting group will vary depending on the eukaryotic organisms. For example , a non - eukaryotic organ - type of chemically reactive group being protected . For ism can belong to the Eubacteria (including but not limited example , if the chemically reactive group is an amine or a to , Escherichia coli, Thermus thermophilus, Bacillus stearo hydrazide, the protecting group can be selected from the thermophilus, Pseudomonas fluorescens, Pseudomonas group of tert -butyloxycarbonyl (t - Boc ) and 9 - fluorenyl aeruginosa , Pseudomonas putida , etc . ) phylogenetic 25 methoxycarbonyl (Fmoc ). If the chemically reactive group domain , or the Archaea ( including but not limited to , Metha is a thiol, the protecting group can be orthopyridyldisulfide . nococcus jannaschii, Methanobacterium thermoautotrophi- If the chemically reactive group is a carboxylic acid , such as cum , Halobacterium such as Haloferax volcanii and butanoic or propionic acid , or a hydroxyl group , the pro Halobacterium species NRC -1 , Archaeoglobus fulgidus, tecting group can be benzyl or an alkyl group such as Pyrococcus furiosus, Pyrococcus horikoshii , Aeuropyrum 30 methyl, ethyl, or tert -butyl . Other protecting groups known pernix , etc . ) phylogenetic domain . in the art may also be used in or with the methods and The term “ subject” as used herein , refers to an animal, in compositions described herein , including photolabile groups some embodiments a mammal, and in other embodiments a such as Nvoc and MeNvoc . Other protecting groups known human , who is the object of treatment, observation or in the art may also be used in or with the methods and experiment. An animal may be a companion animal ( e . g ., 35 compositions described herein . dogs , cats , and the like ), farm animal (e .g ., cows, sheep , By way of example only , blocking /protecting groups may pigs , horses, and the like ) or a laboratory animal ( e . g ., rats , be selected from : mice , guinea pigs , and the like ). The term “ effective amount” as used herein refers to that amount of the modified non -natural amino acid polypeptide 40 being administered which will relieve to some extent one or more of the symptoms of the disease , condition or disorder being treated . Compositions containing the modified non C= natural amino acid polypeptide described herein can be deallyl Bn administered for prophylactic , enhancing , and / or therapeutic 45 treatments . The terms“ enhance ” or “ enhancing ” means to increase or to prolong either in potency or duration a desired effect. Thus , C - o in regard to enhancing the effect of therapeutic agents, the H2 term " enhancing” refers to the ability to increase or prolong , 50 either in potency or duration , the effect of other therapeutic agents on a system . An “ enhancing - effective amount, ” as Cbz alloc used herein , refers to an amount adequate to enhance the effect of another therapeutic agent in a desired system . When HzC H3C (H3C )3C used in a patient, amounts effective for this use will depend 55 Me Et t - butyl on the severity and course of the disease , disorder or HzC CH3 condition , previous therapy, the patient' s health status and response to the drugs , and the judgment of the treating physician . (H3C ) 3C The term “ modified , ” as used herein refers to any changes 60 TBDMS Teoc made to a given polypeptide , such as changes to the length of the polypeptide, the amino acid sequence , chemical structure , co - translationalmodification , or post - translational (CH3 ) modification of a polypeptide . The form “ (modified ) ” term means that the polypeptides being discussed are optionally 65 © HC0H3C07 modified , that is , the polypeptides under discussion can be Boc PMBn modified or unmodified . US 9 , 962, 450 B2 39 40 -continued 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 100 % , more O than 100 % , 150 % ,more than 150 % , 200 % ,more than 200 % . In another embodiment of the present invention is a method (C6H5 ) 3C — HzC of treatment of a patient in need thereof to increase the trityl acetyl 5 patient' s ANP expression by administering a therapeutically effective amount of relaxin polypeptide to increase NO production by 10 % , 15 % , 20 % , 25 % , 30 % , 35 % , 40 % , 45 % , 50 % , 55 % , 60 % , 65 % , 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , H . C 100 % , more than 100 % , 150 % , more than 150 % , 200 % , more than 200 % . Present methods whereby a peg - relaxin of the present invention has a 10 - fold increase in AUC as compared to a wild type relaxin 15 - fold increase ; more than 15 - fold is increase ; 20 - fold increase ; more than 20 - fold increase ; 25 Fmoc fold increase ; more than 25 - fold increase ; 30 - fold increase ; more than 30 - fold increase ; 35 - fold increase ; more than 35 - fold increase; 40 - fold increase ; more than 40 - fold Other protecting groups are described in Greene and increase ; 45 - fold increase ; more than 45 - fold increase ; Wuts , Protective Groups in Organic Synthesis , 3rd Ed ., John1 20 50 -fold increase ; more than 50 - fold increase ; 55 - fold Wiley & Sons, New York , N . Y ., 1999 , which is incorporated increase ; more than 55 -fold increase ; 60 - fold increase ; more herein by reference in its entirety . than 60 - fold increase ; 65 - fold increase; more than 65 - fold In therapeutic applications, compositions containing thethe increase ; 70 - fold increase ; more than 70 - fold increase ; modified non - natural amino acid polypeptide are adminis 75 - fold increase ; more than 75 - fold increase ; 80 - fold tered to a patient already suffering from a disease , condition 25 increase; more than 80 - fold increase ; 85 - fold increase; more or disorder, in an amount sufficient to cure or at least than 85 - fold increase ; 90 - fold increase ; more than 90 - fold partially arrest the symptoms of the disease , disorder or increase ; 95 - fold increase ; more than 95 - fold increase ; 100 condition . Such an amount is defined to be a " therapeutically fold increase ; more than 100 - fold increase. effective amount, ” and will depend on the severity and The term “ treating " is used to refer to either prophylactic course of the disease , disorder or condition , previous 30 and / or therapeutic treatments . therapy, the patient' s health status and response to the drugs, Non -naturally encoded amino acid polypeptides pre and the judgment of the treating physician . It is considered sented herein may include isotopically - labelled compounds well within the skill of the art for one to determine such with one or more atoms replaced by an atom having an therapeutically effective amounts by routine experimenta atomic mass ormass number different from the atomic mass tion ( e . g . , a dose escalation clinical trial) . 35 or mass number usually found in nature . Examples of Relaxin polypeptides of the present invention can be used isotopes that can be incorporated into the present com to modulate vasoconstriction , NO production , ET- 1, Ang II , pounds include isotopes of hydrogen , carbon , nitrogen , and platelet aggregation . In one embodiment of the present oxygen , fluorine and chlorine, such as 2H , 3H , 13C , 14C , invention , a patient in need thereof receives a therapeutic 15N , 180 , 170 , 35S , 18F , 36C1, respectively . Certain iso amount of relaxin polypeptides of the present invention that 40 topically -labelled compounds described herein , for example would decrease the patient' s vasoconstriction over the base those into which radioactive isotopes such as 3H and 14C line of their seeking treatment by 10 % , 15 % , 20 % , 25 % , are incorporated , may be useful in drug and / or substrate 30 % , 35 % , 40 % , 45 % , 50 % , 55 % , 60 % , 65 % , 70 % , 75 % , tissue distribution assays. Further , substitution with isotopes 80 % , 85 % , 90 % , 95 % , 100 % , more than 100 % , 150 % , more such as deuterium , i . e ., 2H , can afford certain therapeutic than 150 % , 200 % , more than 200 % . In another embodiment 45 advantages resulting from greater metabolic stability , for of the present invention is a method of treatment of a patient example increased in vivo half - life or reduced dosage in need thereof to increase the patient' s NO production by requirements . administering a therapeutically effective amount of relaxin All isomers including but not limited to diastereomers , polypeptide to increase NO production by 10 % , 15 % , 20 % , enantiomers , and mixtures thereof are considered as part of 25 % , 30 % , 35 % , 40 % , 45 % , 50 % , 55 % , 60 % , 65 % , 70 % , 50 the compositions described herein . In additional or further 75 % , 80 % , 85 % , 90 % , 95 % , 100 % , more than 100 % , 150 % , embodiments , the non -naturally encoded amino acid poly more than 150 % , 200 % , more than 200 % . peptides are metabolized upon administration to an organ In one embodiment of the present invention is a method ism in need to produce a metabolite that is then used to of treatment of a patient in need thereof with a therapeutic produce a desired effect, including a desired therapeutic amount of relaxin polypeptides of the present invention that 55 effect. In further or additional embodiments are active decreases the patient ' s platelet aggregation by 10 % , 15 % , metabolites of non -naturally encoded amino acid polypep 20 % , 25 % , 30 % , 35 % , 40 % , 45 % , 50 % , 55 % , 60 % , 65 % , tides. 70 % , 75 % , 80 % , 85 % , 90 % , 95 % , 100 % , more than 100 % , In some situations , non - naturally encoded amino acid 150 % , more than 150 % , 200 % , more than 200 % . In another polypeptides may exist as tautomers . In addition , the non embodiment of the present invention is a method of treat- 60 naturally encoded amino acid polypeptides described herein ment of a patient in need thereof with a therapeutic amount can exist in unsolvated as well as solvated forms with of relaxin polypeptides to decrease hypertrophy. In another pharmaceutically acceptable solvents such as water, ethanol, embodiment of the present invention is a method of treat - and the like . The solvated forms are also considered to be ment of a patient in need thereof with a therapeutic amount disclosed herein . Those of ordinary skill in the art will of relaxin polypeptides of the present invention that 65 recognize that some of the compounds herein can exist in decreases the patient ' s CF - stimulated protein synthesis by several tautomeric forms. All such tautomeric forms are 10 % , 15 % , 20 % , 25 % , 300 % , 35 % , 40 % , 45 % , 50 % , 55 % , considered as part of the compositions described herein . US 9 , 962 , 450 B2 41 42 Unless otherwise indicated , conventional methods of In certain embodiments , the protein includes at least one mass spectroscopy, NMR , HPLC , protein chemistry , bio post - translational modification that is made in vivo by one chemistry, recombinant DNA techniques and pharmacology , host cell , where the post- translational modification is not within the skill of the art are employed . normally made by another host cell type. In certain embodi 5 ments , the protein includes at least one post- translational DETAILED DESCRIPTION modification that is made in vivo by a eukaryotic cell, where the post- translational modification is not normally made by I. Introduction a non -eukaryotic cell. Examples of post -translational modi fications include , but are not limited to , glycosylation , Relaxin polypeptides comprising at least one unnatural 10 acetylation , acylation , lipid -modification , palmitoylation , palmitate addition , phosphorylation , glycolipid - linkage amino acid are provided in the invention . In certain embodi modification , and the like. ments of the invention , the relaxin polypeptide with at least In some embodiments , the relaxin polypeptide comprises one unnatural amino acid includes at least one post -trans one or more non -naturally encoded amino acids for glyco lational modification . In one embodiment, the at least one 15 sylation , acetylation , acylation , lipid -modification , palmi post- translational modification comprises attachment of a toylation , palmitate addition , phosphorylation , or glyco molecule including but not limited to , a label, a dye, a lipid - linkage modification of the polypeptide. In some polymer , a water- soluble polymer, a derivative of polyeth embodiments , the relaxin polypeptide comprises one or ylene glycol, a photocrosslinker, a radionuclide , a cytotoxic more non - naturally encoded amino acids for glycosylation compound , a drug , an aflinity label, a photoaffinity label, a 20 of the polypeptide . In some embodiments , the relaxin poly reactive compound, a resin , a second protein or polypeptide peptide comprises one or more naturally encoded amino or polypeptide analog , an antibody or antibody fragment, a acids for glycosylation , acetylation , acylation , lipid -modifi metal chelator, a cofactor, a fatty acid , a carbohydrate , a cation , palmitoylation , palmitate addition , phosphorylation , polynucleotide , a DNA , a RNA , an antisense polynucle or glycolipid - linkage modification of the polypeptide . In otide , a saccharide , a water - soluble dendrimer , a cyclodex - 25 some embodiments , the relaxin polypeptide comprises one trin , an inhibitory ribonucleic acid , a biomaterial, a nano - or more naturally encoded amino acids for glycosylation of particle, a spin label, a fluorophore, a metal- containing the polypeptide . moiety , a radioactive moiety , a novel functional group , a In some embodiments , the relaxin polypeptide comprises group that covalently or noncovalently interacts with other one or more non -naturally encoded amino acid additions molecules , a photocaged moiety , an actinic radiation excit - 30 and / or substitutions that enhance glycosylation of the poly able moiety , a photoisomerizable moiety , biotin , a derivative peptide . In some embodiments , the relaxin polypeptide of biotin , a biotin analogue, a moiety incorporating a heavy comprises one or more deletions that enhance glycosylation atom , a chemically cleavable group , a photocleavable group of the polypeptide . In some embodiments, the relaxin poly an elongated side chain , a carbon -linked sugar, a redox peptide comprises one or more non -naturally encoded amino active agent, an amino thioacid , a toxic moiety , an isotopi- 35 acid additions and / or substitutions that enhance glycosy cally labeled moiety , a biophysical probe, a phosphorescent lation at a different amino acid in the polypeptide. In some group , a chemiluminescent group , an electron dense group , embodiments , the relaxin polypeptide comprises one or a magnetic group , an intercalating group , a chromophore , an more deletions that enhance glycosylation at a different energy transfer agent, a biologically active agent , a detect amino acid in the polypeptide . In some embodiments , the able label , a small molecule , a quantum dot, a nanotrans - 40 relaxin polypeptide comprises one or more non - naturally mitter, a radionucleotide , a radiotransmitter, a neutron - cap - encoded amino acid additions and /or substitutions that ture agent, or any combination of the above or any other enhance glycosylation at a non -naturally encoded amino desirable compound or substance , comprising a second acid in the polypeptide . In some embodiments , the relaxin reactive group to at least one unnatural amino acid com polypeptide comprises one or more non -naturally encoded prising a first reactive group utilizing chemistry methodol- 45 amino acid additions and /or substitutions that enhance gly ogy that is known to one of ordinary skill in the art to be cosylation at a naturally encoded amino acid in the poly suitable for the particular reactive groups. For example , the peptide . In some embodiments , the relaxin polypeptide first reactive group is an alkynyl moiety (including but not comprises one or more naturally encoded amino acid addi limited to , in the unnatural amino acid p - propargyloxyphe - tions and /or substitutions that enhance glycosylation at a nylalanine , where the propargyl group is also sometimes 50 different amino acid in the polypeptide . In some embodi referred to as an acetylene moiety ) and the second reactive ments , the relaxin polypeptide comprises one or more non group is an azido moiety , and [ 3 + 2 ] cycloaddition chemistry naturally encoded amino acid additions and / or substitutions methodologies are utilized . In another example , the first that enhance glycosylation at a naturally encoded amino acid reactive group is the azido moiety (including but not limited in the polypeptide. In some embodiments , the relaxin poly to , in the unnatural amino acid p -azido - L - phenylalanine ) 55 peptide comprises one ormore non - naturally encoded amino and the second reactive group is the alkynyl moiety . In acid additions and / or substitutions that enhance glycosy certain embodiments of the modified relaxin polypeptide of lation at a non - naturally encoded amino acid in the poly the present invention , at least one unnatural amino acid peptide . ( including but not limited to , unnatural amino acid contain - In one embodiment, the post- translational modification ing a keto functional group ) comprising at least one post - 60 comprises attachment of an oligosaccharide to an asparagine translational modification , is used where the at least one by a GlcNAc- asparagine linkage ( including but not limited post - translational modification comprises a saccharide moi- to , where the oligosaccharide comprises (GlcNAc -Man ) 2 ety . In certain embodiments , the post - translational modifi Man -GlcNAc -GlcNAc , and the like ). In another embodi cation is made in vivo in a eukaryotic cell or in a non - ment, the post- translational modification comprises attach eukaryotic cell . A linker, polymer, water soluble polymer , or 65 ment of an oligosaccharide ( including but not limited to , other molecule may attach the molecule to the polypeptide . Gal- GalNAc , Gal- GlcNAc , etc . ) to a serine or threonine by The molecule may be linked directly to the polypeptide. a GalNAc -serine , a GalNAc - threonine , a GlcNAc- serine, or US 9 , 962, 450 B2 43 44 a GlcNAc- threonine linkage. In certain embodiments , a high selectivity . The reaction can be carried out at room protein or polypeptide of the invention can comprise a temperature in aqueous conditions with excellent regiose secretion or localization sequence , an epitope tag , a FLAG lectivity ( 1 , 4 > 1 , 5 ) by the addition of catalytic amounts of tag , a polyhistidine tag , a GST fusion , and /or the like . Cu (I ) salts to the reaction mixture. See, e . g . , Tornoe , et al . , Examples of secretion signal sequences include , but are not 5 ( 2002 ) J . Org . Chem . 67 : 3057 - 3064 ; and , Rostovtsev , et al. , limited to , a prokaryotic secretion signal sequence, a eukary ( 2002 ) Angew . Chem . Int. Ed . 41 :2596 - 2599 ; and WO otic secretion signal sequence , a eukaryotic secretion signal 03 / 101972 . A molecule that can be added to a protein of the sequence 5 '- optimized for bacterial expression , a novel invention through a [3 + 2 ] cycloaddition includes virtually secretion signal sequence , pectate lyase secretion signal any molecule with a suitable functional group or substituent sequence , Omp A secretion signal sequence , and a phage 10 including but not limited to an azido or acetylene derivative . secretion signal sequence . Examples of secretion signal These molecules can be added to an unnatural amino acid sequences, include , but are not limited to , STII (prokary - with an acetylene group , including but not limited to , otic ), Fd GIII and M13 ( phage ), Bg12 ( yeast ), and the signal p - propargyloxyphenylalanine , or azido group , including but sequence bla derived from a transposon . Any such sequence not limited to p - azido -phenylalanine , respectively . may be modified to provide a desired result with the poly - 15 The five -membered ring that results from the Huisgen peptide , including but not limited to , substituting one signal ( 3 + 2 ] cycloaddition is not generally reversible in reducing sequence with a different signal sequence , substituting a environments and is stable against hydrolysis for extended leader sequence with a different leader sequence , etc . periods in aqueous environments . Consequently , the physi The protein or polypeptide of interest can contain at least cal and chemical characteristics of a wide variety of sub one , at least two , at least three , at least four, at least five , at 20 stances can be modified under demanding aqueous condi least six , at least seven , at least eight, at least nine , or ten or tions with the active PEG derivatives of the present more unnatural amino acids . The unnatural amino acids can invention . Even more importantly , because the azide and be the same or different , for example , there can be 1 , 2 , 3 , acetylene moieties are specific for one another ( and do not, 4 , 5 , 6 , 7 , 8 , 9 , 10 or more different sites in the protein that for example , react with any of the 20 common , genetically comprise 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 or more different 25 encoded amino acids) , proteins can be modified in one or unnatural amino acids. In certain embodiments, at least one more specific sites with extremely high selectivity . but fewer than all , of a particular amino acid present in a The invention also provides water soluble and hydrolyti naturally occurring version of the protein is substituted with cally stable derivatives of PEG derivatives and related an unnatural amino acid . hydrophilic polymers having one or more acetylene or azide The present invention provides methods and composi - 30 moieties . The PEG polymer derivatives that contain acety tions based on relaxin comprising at least one non -naturally l ene moieties are highly selective for coupling with azide encoded amino acid . Introduction of at least one non moieties that have been introduced selectively into proteins naturally encoded amino acid into relaxin can allow for the in response to a selector codon . Similarly , PEG polymer application of conjugation chemistries that involve specific derivatives that contain azide moieties are highly selective chemical reactions, including, but not limited to , with one or 35 for coupling with acetylene moieties that have been intro more non -naturally encoded amino acids while not reacting duced selectively into proteins in response to a selector with the commonly occurring 20 amino acids . In some codon . embodiments , relaxin comprising the non - naturally encoded More specifically , the azide moieties comprise , but are not amino acid is linked to a water soluble polymer , such as limited to , alkyl azides , aryl azides and derivatives of these polyethylene glycol (PEG ) , via the side chain of the non - 40 azides . The derivatives of the alkyl and aryl azides can naturally encoded amino acid . This invention provides a include other substituents so long as the acetylene -specific highly efficient method for the selective modification of reactivity is maintained . The acetylene moieties comprise proteins with PEG derivatives, which involves the selective alkyl and aryl acetylenes and derivatives of each . The incorporation of non - genetically encoded amino acids, derivatives of the alkyl and aryl acetylenes can include other including but not limited to , those amino acids containing 45 substituents so long as the azide- specific reactivity is main functional groups or substituents not found in the 20 natu - tained . rally incorporated amino acids, including but not limited to The present invention provides conjugates of substances a ketone , an azide or acetylene moiety , into proteins in having a wide variety of functional groups , substituents or response to a selector codon and the subsequent modifica moieties , with other substances including but not limited to tion of those amino acids with a suitably reactive PEG 50 a label; a dye ; a polymer ; a water -soluble polymer , a derivative . Once incorporated , the amino acid side chains derivative of polyethylene glycol; a photocrosslinker; a can then be modified by utilizing chemistry methodologies radionuclide ; a cytotoxic compound ; a drug ; an affinity known to those of ordinary skill in the art to be suitable for label; a photoaffinity label; a reactive compound ; a resin ; a the particular functional groups or substituents present in the second protein or polypeptide or polypeptide analog ; an non - naturally encoded amino acid . Known chemistry meth - 55 antibody or antibody fragment; a metal chelator; a cofactor, odologies of a wide variety are suitable for use in the present a fatty acid ; a carbohydrate ; a polynucleotide ; a DNA , a invention to incorporate a water soluble polymer into the RNA ; an antisense polynucleotide; a saccharide ; a water protein . Such methodologies include but are not limited to a soluble dendrimer, a cyclodextrin ; an inhibitory ribonucleic Huisgen [ 3 + 2 ] cycloaddition reaction ( see , e . g. , Padwa, A . in acid ; a biomaterial; a nanoparticle ; a spin label ; a fluoro Comprehensive Organic Synthesis, Vol. 4 , ( 1991 ) Ed . Trost, 60 phore , a metal- containing moiety ; a radioactive moiety ; a B . M ., Pergamon , Oxford , p . 1069 - 1109 ; and , Huisgen , R . in novel functional group ; a group that covalently or nonco 1 ,3 - Dipolar Cycloaddition Chemistry , ( 1984 ) Ed . Padwa , A ., valently interacts with other molecules; a photocaged moi Wiley , New York , p . 1 - 176 ) with , including but not limited ety ; an actinic radiation excitable moiety ; a photoisomeriz to , acetylene or azide derivatives , respectively . able moiety ; biotin ; a derivative of biotin ; a biotin analogue ; Because the Huisgen [ 3 + 2 ] cycloaddition method 65 a moiety incorporating a heavy atom ; a chemically cleavable involves a cycloaddition rather than a nucleophilic substi - group ; a photocleavable group ; an elongated side chain ; a tution reaction , proteins can be modified with extremely carbon - linked sugar, a redox - active agent; an amino thio US 9 , 962 , 450 B2 45 46 acid ; a toxic moiety ; an isotopically labeled moiety ; a More specifically , in the case of the acetylene -containing biophysical probe ; a phosphorescent group ; a chemilumi- PEG derivative , a water soluble polymer having at least one nescent group ; an electron dense group ; a magnetic group ; active hydroxyl moiety undergoes a reaction to displace a an intercalating group ; a chromophore ; an energy transfer halogen or other activated leaving group from a precursor agent ; a biologically active agent ; a detectable label; a small 5 that contains an acetylene moiety . Alternatively , a water molecule ; a quantum dot ; a nanotransmitter ; a radionucle - soluble polymer having at least one active nucleophilic or otide ; a radiotransmitter, a neutron - capture agent; or any electrophilic moiety undergoes a reaction with a linking combination of the above , or any other desirable compound agent that has an acetylene at one terminus so that a covalent or substance . The present invention also includes conjugates bond is formed between the PEG polymer and the linking of substances having azide or acetylene moieties with PEG 10 agent and the acetylenemoiety is positioned at the terminus polymer derivatives having the corresponding acetylene or of the polymer. The use of halogen moieties , activated azide moieties . For example , a PEG polymer containing an leaving group , nucleophilic and electrophilic moieties in the azide moiety can be coupled to a biologically active mol- context of organic synthesis and the preparation and use of ecule at a position in the protein that contains a non - PEG derivatives is well established to practitioners in the art. genetically encoded amino acid bearing an acetylene func- 15 The invention also provides a method for the selective tionality . The linkage by which the PEG and the biologically modification of proteins to add other substances to the active molecule are coupled includes but is not limited to the modified protein , including but not limited to water soluble Huisgen [3 + 2 ] cycloaddition product. polymers such as PEG and PEG derivatives containing an It is well established in the art that PEG can be used to azide or acetylene moiety . The azide - and acetylene - con modify the surfaces of biomaterials ( see, e . g . , U . S . Pat. No. 20 taining PEG derivatives can be used to modify the properties 6 ,610 ,281 ; Mehvar, R ., J . Pharm Sci. , 3 ( 1 ) : 125 - 136 ( 2000 ) of surfaces and molecules where biocompatibility , stability , which are incorporated by reference herein ) . The invention solubility and lack of immunogenicity are important, while also includes biomaterials comprising a surface having one at the same time providing a more selective means of or more reactive azide or acetylene sites and one or more of attaching the PEG derivatives to proteins than was previ the azide - or acetylene -containing polymers of the invention 25 ously known in the art. coupled to the surface via the Huisgen [ 3 + 2 ] cycloaddition General Recombinant Nucleic Acid Methods for Use with linkage . Biomaterials and other substances can also be the Invention coupled to the azide - or acetylene - activated polymer deriva . In numerous embodiments of the present invention , tives through a linkage other than the azide or acetylene nucleic acids encoding a relaxin polypeptide of interest will linkage , such as through a linkage comprising a carboxylic 30 be isolated , cloned and often altered using recombinant acid , amine , alcohol or thiol moiety , to leave the azide or methods . Such embodiments are used , including but not acetylene moiety available for subsequent reactions. limited to , for protein expression or during the generation of The invention includes a method of synthesizing the variants , derivatives , expression cassettes , or other azide - and acetylene - containing polymers of the invention . sequences derived from a relaxin polypeptide . In some In the case of the azide - containing PEG derivative , the azide 35 embodiments , the sequences encoding the polypeptides of can be bonded directly to a carbon atom of the polymer . the invention are operably linked to a heterologous pro Alternatively , the azide - containing PEG derivative can be moter. prepared by attaching a linking agent that has the azide A nucleotide sequence encoding a relaxin polypeptide moiety at one terminus to a conventional activated polymer comprising a non - naturally encoded amino acid may be so that the resulting polymer has the azide moiety at its 40 synthesized on the basis of the amino acid sequence of the terminus . In the case of the acetylene - containing PEG parent polypeptide , including but not limited to , having the derivative , the acetylene can be bonded directly to a carbon amino acid sequence shown in SEQ ID NO : 1 and SEQ ID atom of the polymer . Alternatively , the acetylene- containing NO : 2 and then changing the nucleotide sequence so as to PEG derivative can be prepared by attaching a linking agent effect introduction ( i. e ., incorporation or substitution ) or that has the acetylene moiety at one terminus to a conven - 45 removal ( i . e . , deletion or substitution of the relevant amino tional activated polymer so that the resulting polymer has acid residue ( s ) . The nucleotide sequence may be conve the acetylene moiety at its terminus . niently modified by site - directed mutagenesis in accordance More specifically , in the case of the azide -containing PEG with conventional methods . Alternatively , the nucleotide derivative , a water soluble polymer having at least one sequence may be prepared by chemical synthesis , including active hydroxyl moiety undergoes a reaction to produce a 50 but not limited to , by using an oligonucleotide synthesizer, substituted polymer having a more reactive moiety, such as wherein oligonucleotides are designed based on the amino a mesylate , tresylate , tosylate or halogen leaving group , acid sequence of the desired polypeptide , and preferably thereon . The preparation and use of PEG derivatives con - selecting those codons that are favored in the host cell in taining sulfonyl acid halides , halogen atoms and other which the recombinant polypeptide will be produced . leaving groups are known to those of ordinary skill in the art . 55 This invention utilizes routine techniques in the field of The resulting substituted polymer then undergoes a reaction recombinant genetics. Basic texts disclosing the general to substitute for the more reactive moiety an azide moiety at methods of use in this invention include Sambrook et al. , the terminus of the polymer . Alternatively , a water soluble Molecular Cloning , A Laboratory Manual (3rd ed . 2001) ; polymer having at least one active nucleophilic or electro - Kriegler , Gene Transfer and Expression : A Laboratory philic moiety undergoes a reaction with a linking agent that 60 Manual ( 1990 ) ; and Current Protocols in Molecular Biology has an azide at one terminus so that a covalent bond is ( Ausubel et al. , eds. , 1994 ) ) . formed between the PEG polymer and the linking agent and General texts which describe molecular biological tech the azide moiety is positioned at the terminus of the polymer . niques include Berger and Kimmel, Guide to Molecular Nucleophilic and electrophilic moieties, including amines , Cloning Techniques , Methods in Enzymology volume 152 thiols, hydrazides , hydrazines , alcohols , carboxylates, alde - 65 Academic Press , Inc. , San Diego , Calif . ( Berger ); Sambrook hydes, ketones , thioesters and the like , are known to those of et al. , Molecular Cloning A Laboratory Manual (2nd Ed. ) , ordinary skill in the art . Vol . 1- 3 , Cold Spring Harbor Laboratory, Cold Spring US 9 , 962 , 450 B2 47 48 Harbor, N . Y . , 1989 (“ Sambrook ” ) and Current Protocols in in Enzymol. 154 : 329 -350 ( 1987) ; Taylor et al. , The use of Molecular Biology , F . M . Ausubel et al. , eds. , Current phosphorothioate -modified DNA in restriction enzyme reac Protocols , a joint venture between Greene Publishing Asso tions to prepare nicked DNA , Nucl . Acids Res . 13 : 8749 ciates, Inc . and John Wiley & Sons, Inc . , ( supplemented 8764 ( 1985 ) ; Taylor et al . , The rapid generation of oligo through 1999 ) ( “ Ausubel" ) ) . These texts describe mutagen - 5 nucleotide - directed mutations at high frequency using esis , the use of vectors, promoters and many other relevant phosphorothioate -modified DNA , Nucl. Acids Res . 13 : topics related to , including but not limited to , the generation 8765 -8785 (1985 ) ; Nakamaye & Eckstein , Inhibition of of genes or polynucleotides that include selector codons for restriction endonuclease Nci I cleavage by phosphorothioate production of proteins that include unnatural amino acids , groups and its application to oligonucleotide - directed muta orthogonal tRNAs, orthogonal synthetases , and pairs 10 genesis , Nucl. Acids Res . 14 : 9679 - 9698 ( 1986 ) ; Sayers et thereof. al. , 5 - 3 ' Exonucleases in phosphorothioate - based oligo Various types ofmutagenesis are used in the invention for nucleotide -directed mutagenesis, Nucl . Acids Res. 16 :791 a variety of purposes , including but not limited to , to 802 ( 1988 ) ; Sayers et al. , Strand specific cleavage of phos produce novel synthetases or tRNAs, to mutate tRNA mol phorothioate - containing DNA by reaction with restriction ecules, to mutate polynucleotides encoding synthetases, to 15 endonucleases in the presence of ethidium bromide , ( 1988 ) produce libraries of tRNAs, to produce libraries of syn Nucl . Acids Res. 16 : 803 -814 ; Kramer et al. , The gapped thetases, to produce selector codons , to insert selector duplex DNA approach to oligonucleotide - directed mutation codons that encode unnatural amino acids in a protein or construction , Nucl. Acids Res . 12 : 9441 - 9456 (1984 ) ; polypeptide of interest. They include but are not limited to Kramer & Fritz Oligonucleotide -directed construction of site -directed , random point mutagenesis , homologous 20 mutations via gapped duplex DNA , Methods in Enzymol. recombination , DNA shuffling or other recursive mutagen - 154 : 350 - 367 ( 1987 ) ; Kramer et al. , Improved enzymatic in esis methods , chimeric construction , mutagenesis using ura - vitro reactions in the gapped duplex DNA approach to cil containing templates, oligonucleotide - directed mutagen - oligonucleotide - directed construction of mutations, Nucl. esis , phosphorothioate -modified DNA mutagenesis , Acids Res. 16 : 7207 ( 1988 ); Fritz et al. , Oligonucleotide mutagenesis using gapped duplex DNA or the like , PCT- 25 directed construction of mutations : a gapped duplex DNA mediated mutagenesis , or any combination thereof. Addi- procedure without enzymatic reactions in vitro , Nucl. Acids tional suitable methods include pointmismatch repair , muta - Res . 16 : 6987 -6999 ( 1988 ) ; Kramer et al. , Different base ! genesis using repair - deficient host strains , restriction base mismatches are corrected with different efficiencies by selection and restriction -purification , deletion mutagenesis , themethyl - directed DNA mismatch - repair system of E . coli , mutagenesis by total gene synthesis , double - strand break 30 Cell 38 :879 - 887 ( 1984 ) ; Carter et al. , Improved oligonucle repair , and the like . Mutagenesis , including but not limited otide site - directed mutagenesis using M13 vectors , Nucl. to , involving chimeric constructs , are also included in the Acids Res. 13 : 4431 -4443 ( 1985 ) ; Carter , Improved oligo present invention . In one embodiment, mutagenesis can be nucleotide -directed mutagenesis using M13 vectors , Meth guided by known information of the naturally occurring ods in Enzymol. 154 : 382 -403 ( 1987 ) ; Eghtedarzadeh & molecule or altered or mutated naturally occurring molecule , 35 Henikoff , Use of oligonucleotides to generate large dele including but not limited to , sequence , sequence compari - tions, Nucl. Acids Res . 14 : 5115 ( 1986 ) ; Wells et al. , sons , physical properties , secondary , tertiary , or quaternary Importance of hydrogen -bond formation in stabilizing the structure , crystal structure or the like . transition state of subtilisin , Phil. Trans . R . Soc . Lond . A The texts and examples found herein describe these 317 : 415 - 423 ( 1986 ) ; Nambiar et al ., Total synthesis and procedures. Additional information is found in the following 40 cloning of a gene coding for the ribonuclease S protein , publications and references cited within : Ling et al. , Science 223 : 1299 - 1301 ( 1984 ) ; Sakmar and Khorana , Total Approaches to DNA mutagenesis : an overview , Anal Bio - synthesis and expression of a gene for the alpha -subunit of chem . 254 ( 2 ): 157 - 178 (1997 ); Dale et al. , Oligonucleotide - bovine rod outer segment guanine nucleotide- binding pro directed random mutagenesis using the phosphorothioate tein ( transducin ) , Nucl. Acids Res. 14 : 6361- 6372 ( 1988 ) ; method , Methods Mol . Biol. 57 : 369 -374 ( 1996 ); Smith , In 45 Wells et al ., Cassette mutagenesis : an efficient method for vitro mutagenesis , Ann . Rev. Genet . 19 :423 -462 ( 1985 ) ; generation of multiple mutations at defined sites, Gene Botstein & Shortle , Strategies and applications of in vitro 34 :315 - 323 ( 1985 ) ; Grundström et al. , Oligonucleotide mutagenesis , Science 229 : 1193 - 1201 ( 1985 ); Carter, Site directed mutagenesis by microscale ‘ shot- gun ’ gene synthe directed mutagenesis , Biochem . J . 237 : 1 - 7 ( 1986 ) ; Kunkel , sis , Nucl. Acids Res. 13 : 3305 - 3316 ( 1985 ) ; Mandecki, The efficiency of oligonucleotide directed mutagenesis , in 50 Oligonucleotide - directed double - strand break repair in plas Nucleic Acids & Molecular Biology (Eckstein , F . and Lilley, mids of Escherichia coli : a method for site -specific muta D . M . J . eds. , Springer Verlag , Berlin ) ( 1987) ; Kunkel, genesis , Proc . Natl . Acad . Sci. USA , 83 : 7177 - 7181 ( 1986 ) ; Rapid and efficient site -specific mutagenesis without phe Arnold , Protein engineering for unusual environments, Cur notypic selection , Proc . Natl . Acad . Sci. USA 82 : 488 - 492 rent Opinion in Biotechnology 4 : 450 - 455 ( 1993 ) ; Sieber, et ( 1985 ) ; Kunkel et al. , Rapid and efficient site - specific muta - 55 al. , Nature Biotechnology, 19 :456 - 460 ( 2001) ; W . P . C . genesis without phenotypic selection , Methods in Enzymol. Stemmer, Nature 370 , 389- 91 ( 1994 ) ; and , I . A . Lorimer, I . 154 , 367 - 382 ( 1987 ) ; Bass et al. , Mutant Trp repressors with Pastan , Nucleic Acids Res. 23 , 3067- 8 ( 1995 ) . Additional new DNA -binding specificities, Science 242 :240 - 245 details on many of the above methods can be found in ( 1988 ) ; Zoller & Smith , Oligonucleotide - directed mutagen Methods in Enzymology Volume 154, which also describes esis using M13 - derived vectors : an efficient and general 60 useful controls for trouble - shooting problems with various procedure for the production of pointmutations in any DNA mutagenesis methods. fragment, Nucleic Acids Res. 10 :6487 -6500 ( 1982 ) ; Zoller Oligonucleotides , e . g ., for use in mutagenesis of the & Smith , Oligonucleotide - directed mutagenesis of DNA present invention , e . g . , mutating libraries of synthetases, or fragments cloned into M13 vectors , Methods in Enzymol. altering tRNAs, are typically synthesized chemically 100 : 468- 500 (1983 ) ; Zoller & Smith , Oligonucleotide - di- 65 according to the solid phase phosphoramidite triester rected mutagenesis : a simple method using two oligonucle - method described by Beaucage and Caruthers , Tetrahedron otide primers and a single - stranded DNA template , Methods Letts . 22 ( 20 ): 1859 - 1862 , ( 1981 ) e .g ., using an automated US 9 , 962 ,450 B2 49 50 synthesizer , as described in Needham - VanDevanter et al ., methods known in the art ( see, for instance, Sambrook ) . In Nucleic Acids Res . , 12 :6159 -6168 ( 1984 ) . addition , kits are commercially available for the purification The invention also relates to eukaryotic host cells , non of plasmids from bacteria , ( see , e .g . , EasyPrepTM , Flex eukaryotic host cells , and organisms for the in vivo incor iPrepTM , both from Pharmacia Biotech ; StrataCleanTM from poration of an unnatural amino acid via orthogonal tRNA ) 5 Stratagene ; and , QIAprepTM from Qiagen ) . The isolated and RS pairs . Host cells are genetically engineered ( including purified plasmids are then further manipulated to produce but not limited to , transformed , transduced or transfected ) other plasmids, used to transfect cells or incorporated into with the polynucleotides of the invention or constructs related vectors to infect organisms. Typical vectors contain which include a polynucleotide of the invention , including transcription and translation terminators , transcription and but not limited to , a vector of the invention , which can be , 10 translation initiation sequences , and promoters useful for for example , a cloning vector or an expression vector . For regulation of the expression of the particular target nucleic example , the coding regions for the orthogonal tRNA , the acid . The vectors optionally comprise generic expression orthogonal tRNA synthetase , and the protein to be deriva - cassettes containing at least one independent terminator tized are operably linked to gene expression control ele sequence , sequences permitting replication of the cassette in ments that are functional in the desired host cell . The vector 15 eukaryotes , or prokaryotes , or both , including but not can be , for example , in the form of a plasmid , a cosmid , a limited to , shuttle vectors ) and selection markers for both phage , a bacterium , a virus , a naked polynucleotide, or a prokaryotic and eukaryotic systems. Vectors are suitable for conjugated polynucleotide . The vectors are introduced into replication and integration in prokaryotes , eukaryotes , or cells and / or microorganisms by standard methods including both . See , Gillam & Smith , Gene 8 : 81 ( 1979 ) ; Roberts , et electroporation (Fromm et al. , Proc . Natl . Acad . Sci. USA 20 al. , Nature , 328 : 731 ( 1987 ) ; Schneider, E ., et al ., Protein 82 , 5824 ( 1985 ) ) , infection by viral vectors , high velocity Expr . Purif . 6 ( 1 ) : 10 - 14 ( 1995 ) ; Ausubel, Sambrook , Berger ballistic penetration by small particles with the nucleic acid (all supra ) . A catalogue of bacteria and bacteriophages useful either within the matrix of small beads or particles, or on the for cloning is provided , e . g ., by the ATCC , e . g ., The ATCC surface (Klein et al. , Nature 327 , 70 -73 ( 1987 ) ), and / or the Catalogue of Bacteria and Bacteriophage ( 1992 ) Gherna et like . Techniques suitable for the transfer of nucleic acid into 25 al. ( eds) published by the ATCC . Additional basic proce cells in vitro include the use of liposomes, microinjection , dures for sequencing , cloning and other aspects ofmolecular cell fusion , DEAE - dextran , the calcium phosphate precipi- biology and underlying theoretical considerations are also tation method , etc . In vivo gene transfer techniques include , found in Watson et al . ( 1992 ) Recombinant DNA Second but are not limited to , transfection with viral ( typically Edition Scientific American Books , NY . In addition , essen retroviral ) vectors and viral coat protein - liposomemediated 30 tially any nucleic acid (and virtually any labeled nucleic transfection [Dzau et al. , Trends in Biotechnology 11 : 205 - acid , whether standard or non - standard ) can be custom or 210 ( 1993 ) ] . In some situations it may be desirable to standard ordered from any of a variety of commercial provide the nucleic acid source with an agent that targets the sources , such as the Midland Certified Reagent Company target cells , such as an antibody specific for a cell surface (Midland , Tex . available on the World Wide Web at mcrc membrane protein or the target cell , a ligand for a receptor 35 .com ) , The Great American Gene Company (Ramona , Calif . on the target cell , etc . Where liposomes are employed , available on the World Wide Web at genco . com ) , Express proteins which bind to a cell surface membrane protein Gen Inc . (Chicago , Ill . available on the World Wide Web at associated with endocytosis may be used for targeting and / or expressgen . com ) , Operon Technologies Inc . ( Alameda , to facilitate uptake , e . g . capsid proteins or fragments thereof Calif . ) and many others . tropic for a particular cell type , antibodies for proteins which 40 Selector Codons undergo internalization in cycling , proteins that target intra Selector codons of the invention expand the genetic codon cellular localization and enhance intracellular half - life . framework of protein biosynthetic machinery . For example , The engineered host cells can be cultured in conventional a selector codon includes , but is not limited to , a unique nutrientmedia modified as appropriate for such activities as , three base codon , a nonsense codon , such as a stop codon , for example , screening steps , activating promoters or select- 45 including but not limited to , an amber codon (UAG ) , an ing transformants. These cells can optionally be cultured ochre codon , or an opal codon (UGA ), an unnatural codon , into transgenic organisms. Other useful references , includ - a four or more base codon , a rare codon , or the like . It is ing but not limited to for cell isolation and culture ( e . g . , for readily apparent to those of ordinary skill in the art that there subsequent nucleic acid isolation ) include Freshney ( 1994 ) is a wide range in the number of selector codons that can be Culture of Animal Cells , a Manual of Basic Technique , third 50 introduced into a desired gene or polynucleotide, including edition , Wiley - Liss , New York and the references cited but not limited to , one or more , two or more , three or more , therein ; Payne et al . ( 1992 ) Plant Cell and Tissue Culture in 4 , 5 , 6 , 7 , 8 , 9 , 10 or more in a single polynucleotide Liquid Systems John Wiley & Sons, Inc . New York , N . Y .; encoding at least a portion of the relaxin polypeptide. It is Gamborg and Phillips ( eds . ) ( 1995 ) Plant Cell, Tissue and also readily apparent to those of ordinary skill in the art that Organ Culture ; Fundamental Methods Springer Lab Manual, 55 there is a wide range in the number of selector codons that Springer- Verlag (Berlin Heidelberg New York ) and Atlas can be introduced into a desired gene or polynucleotide , and Parks ( eds . ) The Handbook of Microbiological Media including but not limited to , one or more , two or more, three ( 1993 ) CRC Press, Boca Raton , Fla . or more , 4 , 5 , 6 , 7 , 8 , 9 , 10 or more total found in the A chain Several well -known methods of introducing target nucleic and B chain polynucleotide sequences encoding at least a acids into cells are available , any of which can be used in the 60 portion of the relaxin polypeptide . invention . These include : fusion of the recipient cells with In one embodiment , the methods involve the use of a bacterial protoplasts containing the DNA , electroporation , selector codon that is a stop codon for the incorporation of projectile bombardment, and infection with viral vectors one or more unnatural amino acids in vivo . For example , an ( discussed further, below ) , etc . Bacterial cells can be used to O - RNA is produced that recognizes the stop codon , includ amplify the number of plasmids containing DNA constructs 65 ing but not limited to , UAG , and is aminoacylated by an of this invention . The bacteria are grown to log phase and the O -RS with a desired unnatural amino acid . This O - tRNA is plasmids within the bacteria can be isolated by a variety of not recognized by the naturally occurring host 's aminoacyl US 9 , 962 , 450 B2 51 52 tRNA synthetases. Conventional site -directed mutagenesis Soc ., 121 :34 . CGGG and AGGU were used to simultane can be used to introduce the stop codon , including but not ously incorporate 2 -naphthylalanine and an NBD derivative limited to , TAG , at the site of interest in a polypeptide of of lysine into streptavidin in vitro with two chemically interest . See , e . g . , Sayers , J . R ., et al. ( 1988 ) , 5 ' - 3 ' Exonu - acylated frameshift suppressor tRNAs. See , e . g . , Hohsaka et cleases in phosphorothioate -based oligonucleotide -directed 5 al. , ( 1999 ) J. Am . Chem . Soc ., 121 : 12194 . In an in vivo mutagenesis . Nucleic Acids Res , 16 :791 - 802. When the study, Moore et al. examined the ability of tRNALeu deriva O -RS , O -TRNA and the nucleic acid that encodes the poly - tives with NCUA anticodons to suppress UAGN codons (N peptide of interest are combined in vivo , the unnatural amino can be U , A , G , or C ) , and found that the quadruplet UAGA acid is incorporated in response to the UAG codon to give can be decoded by a tRNALeu with a UCUA anticodon with a polypeptide containing the unnatural amino acid at the 10 an efficiency of 13 to 26 % with little decoding in the 0 or - 1 specified position . frame. See , Moore et al . , (2000 ) J . Mol . Biol . , 298 : 195 . In The incorporation of unnatural amino acids in vivo can be one embodiment, extended codons based on rare codons or done without significant perturbation of the eukaryotic host nonsense codons can be used in the present invention , which cell. For example , because the suppression efficiency for the can reduce missense readthrough and frameshift suppression UAG codon depends upon the competition between the 15 at other unwanted sites. O -TRNA , including but not limited to , the amber suppressor For a given system , a selector codon can also include one tRNA , and a eukaryotic release factor ( including but not of the natural three base codons , where the endogenous limited to , eRF ) ( which binds to a stop codon and initiates system does not use ( or rarely uses) the natural base codon . release of the growing peptide from the ribosome) , the For example , this includes a system that is lacking a tRNA suppression efficiency can be modulated by , including but 20 that recognizes the natural three base codon , and / or a system not limited to , increasing the expression level of O - TRNA, where the three base codon is a rare codon . and / or the suppressor tRNA . Selector codons optionally include unnatural base pairs . Unnatural amino acids can also be encoded with rare These unnatural base pairs further expand the existing codons . For example , when the arginine concentration in an genetic alphabet. One extra base pair increases the number in vitro protein synthesis reaction is reduced , the rare 25 of triplet codons from 64 to 125 . Properties of third base arginine codon , AGG , has proven to be efficient for insertion pairs include stable and selective base pairing , efficient of Ala by a synthetic tRNA acylated with alanine . See , e . g ., enzymatic incorporation into DNA with high fidelity by a Ma et al. , Biochemistry , 32 : 7939 ( 1993 ). In this case , the polymerase , and the efficient continued primer extension synthetic tRNA competes with the naturally occurring after synthesis of the nascent unnatural base pair. Descrip tRNAArg , which exists as a minor species in Escherichia 30 tions of unnatural base pairs which can be adapted for coli . Some organisms do not use all triplet codons . An methods and compositions include , e . g ., Hirao , et al. , ( 2002 ) unassigned codon AGA in Micrococcus luteus has been An unnatural base pair for incorporating amino acid ana utilized for insertion of amino acids in an in vitro transcrip - logues into protein , Nature Biotechnology , 20 : 177 - 182 . See , tion / translation extract . See , e . g . , Kowal and Oliver, Nucl. also , Wu , Y ., et al . , (2002 ) J . Am . Chem . Soc . 124 :14626 Acid . Res ., 25 : 4685 ( 1997 ) . Components of the present 35 14630 . Other relevant publications are listed below . invention can be generated to use these rare codons in vivo . For in vivo usage , the unnatural nucleoside is membrane Selector codons also comprise extended codons, includ permeable and is phosphorylated to form the corresponding ing but not limited to , four or more base codons , such as , triphosphate . In addition , the increased genetic information four , five , six or more base codons. Examples of four base is stable and not destroyed by cellular enzymes . Previous codons include, but are not limited to , AGGA , CUAG , 40 efforts by Benner and others took advantage of hydrogen UAGA , CCCU and the like . Examples of five base codons bonding patterns that are different from those in canonical include , but are not limited to , AGGAC , CCCCU , CCCUC , Watson - Crick pairs , the most noteworthy example of which CUAGA , CUACU , UAGGC and the like . A feature of the is the iso - C : iso -G pair. See, e .g . , Switzer et al ., ( 1989 ) J. Am . invention includes using extended codons based on frame - Chem . Soc ., 111 : 8322 ; and Piccirilli et al . , ( 1990 ) Nature , shift suppression . Four or more base codons can insert, 45 343 : 33 ; Kool, ( 2000 ) Curr. Opin . Chem . Biol. , 4 :602 . These including but not limited to , one or multiple unnatural amino bases in general mispair to some degree with natural bases acids into the same protein . For example , in the presence of and cannot be enzymatically replicated . Kool and co -work mutated O - URNAs, including but not limited to , a special ers demonstrated that hydrophobic packing interactions frameshift suppressor tRNAs, with anticodon loops, for between bases can replace hydrogen bonding to drive the example , with at least 8 - 10 nt anticodon loops , the four or 50 formation of base pair. See , Kool, ( 2000 ) Curr . Opin . Chem . more base codon is read as single amino acid . In other Biol. , 4 :602 ; and Guckian and Kool, ( 1998 ) Angew . Chem . embodiments , the anticodon loops can decode , including but Int. Ed . Engl. , 36 , 2825 . In an effort to develop an unnatural not limited to , at least a four -base codon , at least a five - base base pair satisfying all the above requirements , Schultz , codon , or at least a six -base codon or more . Since there are Romesberg and co -workers have systematically synthesized 256 possible four -base codons , multiple unnatural amino 55 and studied a series of unnatural hydrophobic bases. A acids can be encoded in the same cell using a four or more PICS :PICS self - pair is found to be more stable than natural base codon . See , Anderson et al ., ( 2002 ) Exploring the base pairs , and can be efficiently incorporated into DNA by Limits of Codon and Anticodon Size , Chemistry and Biol Klenow fragment of Escherichia coli DNA polymerase I ogy, 9 :237 -244 ; Magliery , (2001 ) Expanding the Genetic (KF ). See , e .g ., McMinn et al. , ( 1999 ) J. Am . Chem . Soc ., Code : Selection of Efficient Suppressors of Four- base 60 121: 11585 -6 ; and Ogawa et al. , ( 2000 ) J . Am . Chem . Soc . , Codons and Identification of “ Shifty ” Four- base Codons 122 :3274 . A 3MN : 3MN self - pair can be synthesized by KF with a Library Approach in Escherichia coli, J. Mol. Biol. with efficiency and selectivity sufficient for biological func 307: 755 -769 . tion . See , e. g ., Ogawa et al ., (2000 ) J. Am . Chem . Soc ., For example , four - base codons have been used to incor - 122 : 8803 . However, both bases act as a chain terminator for porate unnatural amino acids into proteins using in vitro 65 further replication . A mutant DNA polymerase has been biosynthetic methods. See , e . g . , Ma et al. , ( 1993 ) Biochem - recently evolved that can be used to replicate the PICS self istry , 32: 7939 ; and Hohsaka et al. , ( 1999) J . Am . Chem . pair . In addition , a 7AI self pair can be replicated . See, e. g. , US 9 , 962, 450 B2 53 54 Tae et al. , (2001 ) J. Am . Chem . Soc . , 123 :7439 . A novel functional group can be reacted with a polymer ( including metallobase pair , Dipic : Py, has also been developed , which but not limited to , poly ( ethylene glycol ) or, alternatively , a forms a stable pair upon binding Cu ( II ) . See , Meggers et al ., second polypeptide containing an alkyne moiety to form a ( 2000 ) J . Am . Chem . Soc ., 122 : 10714 . Because extended stable conjugate resulting for the selective reaction of the codons and unnatural codons are intrinsically orthogonal to 5 azide and the alkyne functional groups to form a Huisgen natural codons , the methods of the invention can take 13 + 2 ] cycloaddition product. advantage of this property to generate orthogonal tRNAs for The generic structure of an alpha - amino acid is illustrated them . A translational bypassing system can also be used to as follows (Formula I ): incorporate an unnatural amino acid in a desired polypep - 10 tide. In a translational bypassing system , a large sequence is incorporated into a gene but is not translated into protein . I The sequence contains a structure that serves as a cue to induce the ribosome to hop over the sequence and resume H2N COOH translation downstream of the insertion . 15 In certain embodiments , the protein or polypeptide of interest (or portion thereof) in the methods and / or compo - A non -naturally encoded amino acid is typically any sitions of the invention is encoded by a nucleic acid . structure having the above - listed formula wherein the R Typically , the nucleic acid comprises at least one selector group is any substituent other than one used in the twenty codon , at least two selector codons, at least three selector 20 natural amino acids, and may be suitable for use in the codons , at least four selector codons, at least five selector present invention . Because the non -naturally encoded amino codons , at least six selector codons , at least seven selector acids of the invention typically differ from the natural amino codons , at least eight selector codons, at least nine selector acids only in the structure of the side chain , the non codons , ten or more selector codons . naturally encoded amino acids form amide bonds with other Genes coding for proteins or polypeptides of interest can 25 amino acids, including but not limited to , natural or non be mutagenized using methods known to one of ordinary naturally encoded , in the same manner in which they are skill in the art and described herein to include, for example , formed in naturally occurring polypeptides . one or more selector codon for the incorporation of an However , the non -naturally encoded amino acids have unnatural amino acid . For example , a nucleic acid for a side chain groups that distinguish them from the natural protein of interest is mutagenized to include one or more 30 amino acids . For example , R optionally comprises an alkyl - , selector codon , providing for the incorporation of one or a ryl- , acyl- , keto - , azido -, hydroxyl - , hydrazine , cyano -, more unnatural amino acids. The invention includes any halo - , hydrazide , alkenyl, alkynl, ether, thiol, seleno - , sul such variant, including but not limited to , mutant, versions f onyl- , borate , boronate , phospho , phosphono , phosphine , of any protein , for example , including at least one unnatural heterocyclic , enone , imine , aldehyde, ester, thioacid , amino acid . Similarly , the invention also includes corre - 35 hydroxylamine , amino group , or the like or any combination sponding nucleic acids, i . e . , any nucleic acid with one or thereof . Other non - naturally occurring amino acids of inter more selector codon that encodes one or more unnatural est that may be suitable for use in the present invention amino acid . include , but are not limited to , amino acids comprising a Nucleic acid molecules encoding a protein of interest such photoactivatable cross - linker, spin - labeled amino acids , as a relaxin polypeptide may be readily mutated to introduce 40 fluorescent amino acids, metal binding amino acids, metal a cysteine at any desired position of the polypeptide. Cys - containing amino acids , radioactive amino acids, amino teine is widely used to introduce reactive molecules , water acids with novel functional groups , amino acids that cova soluble polymers , proteins, or a wide variety of other lently or noncovalently interact with other molecules , pho molecules , onto a protein of interest . Methods suitable for tocaged and / or photoisomerizable amino acids , amino acids the incorporation of cysteine into a desired position of a 45 comprising biotin or a biotin analogue, glycosylated amino polypeptide are known to those of ordinary skill in the art , acids such as a sugar substituted serine , other carbohydrate such as those described in U . S . Pat. No. 6 ,608 ,183 , which is modified amino acids , keto - containing amino acids , amino incorporated by reference herein , and standard mutagenesis acids comprising polyethylene glycol or polyether , heavy techniques . atom substituted amino acids , chemically cleavable and / or Non - Naturally Encoded Amino Acids 50 photocleavable amino acids , amino acids with an elongated A very wide variety of non - naturally encoded amino acids side chains as compared to natural amino acids , including are suitable for use in the present invention . Any number of but not limited to , polyethers or long chain hydrocarbons , non - naturally encoded amino acids can be introduced into a including but not limited to , greater than about 5 or greater relaxin polypeptide . In general, the introduced non -naturally than about 10 carbons , carbon - linked sugar - containing encoded amino acids are substantially chemically inert 55 amino acids , redox - active amino acids, amino thioacid con toward the 20 common , genetically - encoded amino acids taining amino acids, and amino acids comprising one or ( i. e ., alanine, arginine , asparagine, aspartic acid , cysteine , more toxic moiety . glutamine , glutamic acid , glycine , histidine , isoleucine , leu - Exemplary non -naturally encoded amino acids that may cine, lysine , methionine , phenylalanine , proline , serine , be suitable for use in the present invention and that are threonine, tryptophan , tyrosine , and valine ) . In some 60 useful for reactions with water soluble polymers include , but embodiments , the non -naturally encoded amino acids are not limited to , those with carbonyl, aminooxy , hydrazine , include side chain functional groups that react efficiently and hydrazide , semicarbazide, azide and alkyne reactive groups. selectively with functional groups not found in the 20 In some embodiments , non - naturally encoded amino acids common amino acids (including but not limited to , azido , comprise a saccharide moiety . Examples of such amino ketone , aldehyde and aminooxy groups) to form stable 65 acids include N -acetyl - L - glucosaminyl- L - serine , N - acetyl conjugates . For example , a relaxin polypeptide that includes L - galactosaminyl- L - serine , N - acetyl - L - glucosaminyl- L a non -naturally encoded amino acid containing an azido threonine, N -acetyl - L - glucosaminyl - L -asparagine and US 9 , 962 ,450 B2 55 56 O -mannosaminyl - L - serine . Examples of such amino acids substituted tyrosines, ortho - substituted tyrosines, and meta also include examples where the naturally occurring N - or substituted tyrosines , where the substituted tyrosine com O - linkage between the amino acid and the saccharide is prises, including but not limited to , a keto group ( including replaced by a covalent linkage not commonly found in but not limited to , an acetyl group ) , a benzoyl group , an nature — including but not limited to , an alkene , an oxime, a 5 amino group , a hydrazine , an hydroxyamine , a thiol group , thioether , an amide and the like. Examples of such amino a carboxy group , an isopropyl group , a methyl group , a acids also include saccharides that are not commonly found C6 - C20 straight chain or branched hydrocarbon , a saturated in naturally -occurring proteins such as 2 -deoxy - glucose , or unsaturated hydrocarbon , an O -methyl group , a polyether 2 - deoxygalactose and the like . group , a nitro group , an alkynyl group or the like . In Many of the non -naturally encoded amino acids provided 10 addition , multiply substituted aryl rings are also contem herein are commercially available , e . g . , from Sigma- Aldrich plated . Glutamine analogs that may be suitable for use in the ( St. Louis , Mo. , USA ) , Novabiochem ( a division of EMD present invention include, but are not limited to , a -hydroxy Biosciences , Darmstadt, Germany ) , or Peptech (Burlington , derivatives, g - substituted derivatives , cyclic derivatives , and Mass. , USA ) . Those that are not commercially available are amide substituted glutamine derivatives . Example phenyl optionally synthesized as provided herein or using standard 15 alanine analogs that may be suitable for use in the present methods known to those of ordinary skill in the art . For invention include , but are not limited to , para -substituted organic synthesis techniques, see, e . g ., Organic Chemistry phenylalanines , ortho - substituted phenyalanines, and meta by Fessendon and Fessendon , ( 1982 , Second Edition , Wil substituted phenylalanines , where the substituent comprises, lard Grant Press, Boston Mass . ); Advanced Organic Chem - including but not limited to , a hydroxy group , a methoxy istry by March ( Third Edition , 1985 , Wiley and Sons, New 20 group , a methyl group , an allyl group , an aldehyde , an azido , York ) ; and Advanced Organic Chemistry by Carey and an iodo , a bromo, a keto group ( including but not limited to , Sundberg ( Third Edition , Parts A and B , 1990 , Plenum Press , an acetyl group ) , a benzoyl, an alkynyl group , or the like . New York ). See , also , U . S . Pat. Nos. 7 , 045 , 337 and 7 , 083 , Specific examples of unnatural amino acids that may be 970 , which are incorporated by reference herein . In addition suitable for use in the present invention include , but are not to unnatural amino acids that contain novel side chains, 25 limited to , a p -acetyl - L -phenylalanine , an O -methyl - L - tyro unnatural amino acids that may be suitable for use in the sine, an L - 3 - ( 2 -naphthyl ) alanine , a 3 -methyl - phenylalanine , present invention also optionally comprise modified back - an 0 - 4 - allyl- L - tyrosine, a 4 -propyl - L -tyrosine , a tri - O bone structures, including but not limited to , as illustrated by acetyl- GlcNAc b - serine , an L -Dopa , a fluorinated phenyl the structures of Formula II and III: alanine , an isopropyl- L -phenylalanine , a p - azido - L -phenyl 30 alanine , a p -acyl - L - phenylalanine , a p -benzoyl - L phenylalanine , an L - phosphoserine , a phosphonoserine , a ? phosphonotyrosine, a p - iodo - phenylalanine , a p -bromophe nylalanine , a p -amino - L -phenylalanine , an isopropyl- L -phe nylalanine, and a p -propargyloxy -phenylalanine , and the 2 C — ??YH 35 like . Examples of structures of a variety of unnatural amino acids that may be suitable for use in the present invention are provided in , for example , WO 2002 /085923 entitled “ In vivo RR incorporation of unnatural amino acids. ” See also Kiick et al ., ( 2002 ) Incorporation of azides into recombinant proteins 40 for chemoselective modification by the Staudinger ligation , H2N Co2H PNAS 99: 19 -24 , which is incorporated by reference herein , for additional methionine analogs . International Application wherein Z typically comprises OH , NH2, SH , NH - R ', or No . PCT /US06 / 47822 entitled " Compositions Containing , S — R ' ; X and Y , which can be the same or different, typically Methods Involving , and Uses of Non - natural Amino Acids comprise S or O , and R and R ', which are optionally the 45 and Polypeptides , ” which is incorporated by reference same or different, are typically selected from the same list of herein , describes reductive alkylation of an aromatic amine constituents for the R group described above for the unnatu - moieties , including but not limited to , p - amino - phenylala ral amino acids having Formula I as well as hydrogen . For nine and reductive amination . example , unnatural amino acids of the invention optionally In one embodiment, compositions of relaxin polypeptide comprise substitutions in the amino or carboxyl group as 50 that include an unnatural amino acid ( such as p - (propargy illustrated by Formulas II and III . Unnatural amino acids of loxy ) - phenyalanine ) are provided . Various compositions this type include , but are not limited to , a - hydroxy acids, comprising p - ( propargyloxy ) -phenyalanine and , including a -thioacids , a -aminothiocarboxylates , including but not but not limited to , proteins and /or cells , are also provided . In limited to , with side chains corresponding to the common one aspect, a composition that includes the p - (propargy twenty natural amino acids or unnatural side chains . In 55 loxy ) -phenyalanine unnatural amino acid , further includes addition , substitutions at the a - carbon optionally include , an orthogonal tRNA . The unnatural amino acid can be but are not limited to , L , D , or a - a - disubstituted amino acids bonded ( including but not limited to , covalently ) to the such as D -glutamate , D - alanine , D -methyl - O -tyrosine , orthogonal tRNA , including but not limited to , covalently aminobutyric acid , and the like . Other structural alternatives bonded to the orthogonal tRNA though an amino - acyl bond , include cyclic amino acids, such as proline analogues as well 60 covalently bonded to a 3 'OH or a 2 'OH of a terminal ribose as 3 , 4 , 6 , 7 , 8 , and 9 membered ring proline analogues , ß sugar of the orthogonal tRNA , etc . and y amino acids such as substituted ß - alanine and y -amino The chemical moieties via unnatural amino acids that can butyric acid . be incorporated into proteins offer a variety of advantages Many unnatural amino acids are based on natural amino and manipulations of the protein . For example , the unique acids , such as tyrosine, glutamine , phenylalanine, and the 65 reactivity of a keto functional group allows selective modi like , and are suitable for use in the present invention . fication of proteins with any of a number of hydrazine - or Tyrosine analogs include, but are not limited to , para - hydroxylamine -containing reagents in vitro and in vivo . A US 9 ,962 ,450 B2 57 58 heavy atom unnatural amino acid , for example , can be useful para - acetylphenylalanine amino acid . The synthesis of for phasing X - ray structure data . The site -specific introduc p - acetyl- ( + / - ) -phenylalanine and m - acetyl- ( + / - ) -phenylala tion of heavy atoms using unnatural amino acids also nine are described in Zhang, Z ., et al. , Biochemistry 42 : provides selectivity and flexibility in choosing positions for 6735 -6746 (2003 ) , incorporated by reference. Other carbo heavy atoms. Photoreactive unnatural amino acids ( includ - 5 nyl - or dicarbonyl- containing amino acids can be similarly ing but not limited to , amino acids with benzophenone and prepared by one of ordinary skill in the art. Further, non arylazides ( including but not limited to , phenylazide ) side limiting exemplary syntheses of non - natural amino acid that chains ), for example , allow for efficient in vivo and in vitro are included herein are presented in FIGS . 4 , 24 - 34 and photocrosslinking of protein . Examples of photoreactive 36 - 39 of U . S . Pat. No . 7 ,083 , 970 , which is incorporated by unnatural amino acids include , but are not limited to , 10 reference herein in its entirety . p -azido -phenylalanine and p -benzoyl - phenylalanine . The Amino acids with an electrophilic reactive group allow protein with the photoreactive unnatural amino acids can for a variety of reactions to link molecules via nucleophilic then be crosslinked at will by excitation of the photoreactive addition reactions among others. Such electrophilic reactive group - providing temporal control. In one example , the groups include a carbonyl group ( including a keto group and methyl group of an unnatural amino can be substituted with 15 a dicarbonyl group ) , a carbonyl- like group (which has an isotopically labeled , including but not limited to , methyl reactivity similar to a carbonyl group ( including a keto group , as a probe of local structure and dynamics, including group and a dicarbonyl group ) and is structurally similar to but not limited to , with the use of nuclear magnetic reso a carbonyl group ), a masked carbonyl group (which can be nance and vibrational spectroscopy . Alkynyl or azido func - readily converted into a carbonyl group ( including a keto tional groups, for example , allow the selective modification 20 group and a dicarbonyl group ) ), or a protected carbonyl of proteins with molecules through a [3 + 2 ]cycloaddition group (which has reactivity similar to a carbonyl group reaction . ( including a keto group and a dicarbonyl group ) upon A non - natural amino acid incorporated into a polypeptide deprotection ). Such amino acids include amino acids having at the amino terminus can be composed of an R group that the structure of Formula (IV ) : is any substituent other than one used in the twenty natural 25 ( IV ) amino acids and a 2nd reactive group different from the NH2 R3 group normally present in a -amino acids ( see Formula I ). A R3LA similar non - natural amino acid can be incorporated at the m carboxyl terminus with a 2nd reactive group different from the COOH group normally present in a - amino acids (see 30 R1- Formula I) . The unnatural amino acids of the invention may be RA O selected or designed to provide additional characteristics ununavailable in the twenty natural amino acids . For example , wherein : unnatural amino acid may be optionally designed or selected 355 A is optional, and when present is lower alkylene , substi to modify the biological properties of a protein , e . g. , into tuted lower alkylene, lower cycloalkylene, substituted lower which they are incorporated . For example , the following cycloalkylene, lower alkenylene, substituted lower alk properties may be optionally modified by inclusion of an enylene , alkynylene, lower heteroalkylene, substituted het unnatural amino acid into a protein : toxicity , biodistribution , eroalkylene, lower heterocycloalkylene , substituted lower solubility , stability , e . g . , thermal, hydrolytic , oxidative , 40 heterocycloalkylene , arylene, substituted arylene , het resistance to enzymatic degradation , and the like , facility of eroarylene, substituted heteroarylene , alkarylene , substi purification and processing, structural properties, spectro tuted alkarylene , aralkylene , or substituted aralkylene ; scopic properties , chemical and /or photochemical proper B is optional, and when present is a linker selected from the ties , catalytic activity , redox potential , half -life , ability to group consisting of lower alkylene , substituted lower alky react with other molecules , e . g ., covalently or noncova - 45 leneheteroalkylene , lower alkenylene, substituted , substituted lower lowerheteroalkylene alkenylene. , lowerO lently , and the like . 0 - alkylene or substituted alkylene ) - , - S — , - S - alky Structure and Synthesis of Non - Natural Amino Acids : Car - lene or substituted alkylene )- , - S ( O ), where k is 1 , 2 , or bonyl, Carbonyl- Like , Masked Carbonyl, Protected Carbo - 3 , - S (O ) (alkylene or substituted alkylene )- , - C ( O ) - , nyl Groups , and Hydroxylamine Groups - C ( O ) - (alkylene or substituted alkylene ) -, - C ( S ) , In some embodiments the present invention provides 50 _ C ( S ) - ( alkylene or substituted alkylene ) -, — N ( R ') — , relaxin linked to a water soluble polymer, e . g . , a PEG , by an NR ' - (alkylene or substituted alkylene ) -, - C ( O ) N ( R ') , oxime bond . CON (R ') - ( alkylene or substituted alkylene ) -, CSN Many types of non - naturally encoded amino acids are ( R ') - CSN ( R ') - (alkylene or substituted alkylene )- , suitable for formation of oxime bonds. These include, but - N ( R ' )CO -( alkylene or substituted alkylene ) - , - N ( R ' ) C are not limited to , non -naturally encoded amino acids con - 55 00( - , - S ( O ) kN ( R ' ) - , - N ( R ' ) C ( O ) N ( R ) - , - NRC taining a carbonyl, dicarbonyl , or hydroxylamine group . ( S ) N ( R ') — , - N ( R ') S ( O ) , N ( R ') — , - N ( R ') — N — , - C ( R ') Such amino acids are described in U . S . Patent Publication = N , C ( R ') = N = N ( R ') — , - C ( R ') = N — N — , Nos. 2006 /0194256 , 2006 / 0217532 , and 2006 / 0217289 and - C ( R ') 2 — N — N — , and C ( R ') 2 — N ( R ') — N ( R ') — , WO 2006 / 069246 entitled “ Compositions containing, meth - Jiswhere each R ' is independently H , alkyl , or substituted alkyl; ods involving , and uses of non - natural amino acids and 60 polypeptides, ” which are incorporated herein by reference in their entirety . Non -naturally encoded amino acids are also described in U . S . Pat. No . 7 , 083 , 970 and U . S . Pat. No . m 7 ,045 ,337 , which are incorporated by reference herein in their entirety . 65 Some embodiments of the invention utilize relaxin poly www meno ma man peptides that are substituted at one or more positions with a US 9 , 962 ,450 B2 59 60 - continued lene , lower alkenylene , substituted lower alkenylene, lower R " heteroalkylene , substituted lower heteroalkylene , o , OR " SR " - O - ( alkylene or substituted alkylene ) , S - , - S - ( alky lene or substituted alkylene ) -, - S ( O ) : - where k is 1 , 2 , or m 5 3 , - S ( O ) k (alkylene or substituted alkylene) -, - C ( O ) - , ma mus C ( O )- ( alkylene or substituted alkylene ) , C (S ) , men wanan wan C ( S )- (alkylene or substituted alkylene )- , - N (R ') — , - NR ' - alkylene or substituted alkylene) -, - C ( O ) N ( R ') — , RIN Or CON ( R ') - ( alkylene or substituted alkylene ) -, CSN 10 ( R ' ) , CSN ( R ') - (alkylene or substituted alkylene ) -, mu min www mu — N ( R ') CO -( alkylene or substituted alkylene )- , - N ( R ') C ( 0 ) O - , - S ( O ), N ( R ') — , - N ( R ') C (O ) N ( R ') — , - N ( R ' ) C R is H , alkyl, substituted alkyl, cycloalkyl , or substituted (S )N (R ') — , - N (R ') S (O ) N (R ') — , - N ( R ') — N — , - C ( R ') cycloalkyl; 15 = N — , C ( R ' ) = N — N ( R ') — , - C ( R ' ) = N — N — , each R " is independently H , alkyl, substituted alkyl, or a C ( R ') 2 – N = N — , and C ( R ') 2 - N (R ') — N (R ') — , protecting group , or when more than one R " group is where each R ' is independently H , alkyl , or substituted alkyl; present, two R " optionally form a heterocycloalkyl; R is H , alkyl, substituted alkyl, cycloalkyl, or substituted R , is optional , and when present, is H , an amino protectingdi cycloalkyl; group , resin , amino acid , polypeptide, or polynucleotide ; 20 R , is optional, and when present, is H , an amino protecting and group , resin , amino acid , polypeptide, or polynucleotide ; R , is optional, and when present, is OH , an ester protecting and group , resin , amino acid , polypeptide , or polynucleotide ; R2 is optional , and when present, is OH , an ester protecting each of Rz and R , is independently H , halogen , lower alkyl, group , resin , amino acid , polypeptide , or polynucleotide; or substituted lower alkyl, or Rz and R4 or two Rz groups - optionally form a cycloalkyl or a heterocycloalkyl; that when A is (CH2 )44 B is not — NHC ( O ) or the - A - B - J - R groups together form a bicyclic or tricyclic (CH2CH2 ) — ; and that when A and B are absent, R is not cycloalkyl or heterocycloalkyl comprising at least one car - methyl . .. bonyl group , including a dicarbonyl group , protected car - 30 In addition , amino acids having the structure of Formula bonyl group , including a protected dicarbonyl group , or (VI ) are included : masked carbonyl group , including a masked dicarbonyl group ; or the - J- R group together forms a monocyclic or bicyclic (VI ) cycloalkyl or heterocycloalkyl comprising at least one car Ra bonyl group , including a dicarbonyl group , protected car 35 bonyl group , including a protected dicarbonyl group , or masked carbonyl group , including a masked dicarbonyl group ; with a proviso that when A is phenylene and each R , is H , 10 B is present ; and that when A is – ( CH2) 4 - and each Rz is 40 - H , B is not — NHC ( O ) (CH ,CH , ) ; and that when A and B are absent and each Rz is H , R is not methyl. In addition , having the structure of Formula ( V ) are included : 45 wherein : (V ) B is a linker selected from the group consisting of lower alkylene , substituted lower alkylene, lower alkenylene , sub stituted lower alkenylene, lower heteroalkylene , substituted R , lower heteroalkylene , 0 % , 0 - (alkylene or substituted alkylene) - , - S — , - S - alkylene or substituted alkylene ) - , Ris - S ( O ) - where k is 1 , 2 , or 3 , - S ( O ) k (alkylene or substituted alkylene )- , - C ( O ) , C (O ) -( alkylene or sub 55 stituted alkylene ) , C ( S ) , C ( S ) - (alkylene or substi tuted alkylene ) - , - N ( R ') — , - NR '- ( alkylene or substituted wherein : alkylene )- , - C (O ) N (R ') — , - CON (R ' )- ( alkylene or substi A is optional, and when present is lower alkylene , substi - tuted alkylene ) - , CSN ( R ') — , CSN ( R ' ) - (alkylene or tuted lower alkylene, lower cycloalkylene , substituted lower substituted alkylene ) - , - N ( R ') CO -( alkylene or substituted cycloalkylene , lower alkenylene , substituted lower alk - 60 alkylene ) - , - N ( R ') C ( O ) O - , S ( O ), N ( R ') — , - N ( R ') C enylene , alkynylene , lower heteroalkylene , substituted het - ( O ) N ( R ') — , - N ( R ' ) C ( S ) N ( R ') — , — N ( R ' ) S ( O ) N ( R ') — , eroalkylene , lower heterocycloalkylene , substituted lower — N ( R ' ) — N — , - C ( R ') — N — , C ( R ' ) = N / N ( R ') — , heterocycloalkylene , arylene , substituted arylene , het C ( R ') — N — N — , - C ( R ') 2 — N — N — , and C ( R ') 2 — N eroarylene , substituted heteroarylene , alkarylene , substi - ( R ') — N ( R ' ) — , where each R ' is independently H , alkyl, or tuted alkarylene , aralkylene , or substituted aralkylene ; B is 65 substituted alkyl; optional, and when present is a linker selected from the R is H , alkyl, substituted alkyl , cycloalkyl, or substituted group consisting of lower alkylene , substituted lower alky - cycloalkyl; US 9 , 962 ,450 B2 61 62 R , is optional, and when present, is H , an amino protecting group , resin , amino acid , polypeptide , or polynucleotide ; -continued and R , is optional, and when present, is OH , an ester protecting , and group , resin , amino acid , polypeptide , or polynucleotide ; 5 each Rq is independently selected from the group consisting of H , halogen , alkyl, substituted alkyl, — N (R ') 2, - C ( O ) R ' where k is 1 , 2 , or 3 , C (O ) N (R ') 2 , — OR ', and - S (O ), R ', where each R ' is independently H , alkyl, or substituted alkyl. HN In addition , the following amino acids are included : 10

15

H2N 20 HNH N wherein such compounds are optionally amino protected group , carboxyl protected or a salt thereof. In addition , any 25 of the following non -natural amino acids may be incorpo rated into a non -natural amino acid polypeptide. In addition , the following amino acids having the struc ture of Formula (VII ) are included : H2N COOH 30 (VII ) ( CRa) BR. 35 1 , R2 HNHNX OH

40 wherein B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alky lene, lower alkenylene , substituted lower alkenylene , lower heteroalkylene, substituted lower heteroalkylene , o - , 45 — O - ( alkylene or substituted alkylene ) -, - S — , - S -( alky lene or substituted alkylene )- , - S (O ) - where k is 1, 2 , or H?N 3 , - S ( O ) (alkylene or substituted alkylene ) -, - C ( O ) - , - C (O )- (alkylene or substituted alkylene ) , C ( S ) , C (S )- ( alkylene or substituted alkylene )- , — N (R ') — , 50 — NR '- alkylene or substituted alkylene ) -, - C (O ) N (R ') — , _ CON ( R ' ) - ( alkylene or substituted alkylene ) , CSN NH ( R ') — , CSN ( R ') - ( alkylene or substituted alkylene ) -, hubbybartobe - N (R ') CO -( alkylene or substituted alkylene ) -, - N (R ') C ( 0 ) O , - S ( O ) N ( R ' ) - , - N (R ') C (O ) N (R ') — , - N ( R ') C 55 (S ) N (R ' ) - , - N (R ') S (O ) N (R ') — , - N ( R ') — N — , - C (R ') HAN COOH = N , C ( R ') — N — N ( R ') , C ( R ') — N — N — , C (R ') 2 - N = N — , and C ( R ') 2 - N (R ') — N (R ') — , where each R ' is independently H , alkyl, or substituted alkyl; R is H , alkyl, substituted alkyl, cycloalkyl, or substituted 60 cycloalkyl; R is optional , and when present, is H , an amino protecting group , resin , amino acid , polypeptide , or polynucleotide ; and OH R2 is optional, and when present, is OH , an ester protecting H?N 65 group , resin , amino acid , polypeptide , or polynucleotide ; each R , is independently selected from the group consisting of H , halogen , alkyl , substituted alkyl, - N (R ') 2, - C (O ), R ' US 9 ,962 , 450 B2 63 64 where k is 1, 2 , or 3 , - C ( O )N (R ') 2 , — OR ', and - S (O ) R ', - continued where each R ' is independently H , alkyl , or substituted alkyl; and n is 0 to 8 ; with a proviso that when A is (CH2 ) 4 — , B is not - NHC 5 (O ) (CH2CH2 ) . NH In addition , the following amino acids are included :

10 OH HNOHH N H?NYHUN

?? HN HN

OH 25 HNOHHN H2N OH HON HON

HN

JOH OH OH HN H2N H2N H2N H2NTOH , and 40 wherein such compounds are optionally amino protected , optionally carboxyl protected , optionally amino protected 0= and carboxyl protected , or a salt thereof. In addition , these NH non - natural amino acids and any of the following non 45 natural amino acids may be incorporated into a non -natural amino acid polypeptide . In addition , the following amino acids having the struc OH ture of Formula (VIII ) are included : HN LOH 50 HN ( VIII )

55

60 wherein A is optional, and when present is lower alkylene , substituted lower alkylene, lower cycloalkylene , substituted lower cycloalkylene , lower alkenylcne , substituted lower OH OH alkenylene , alkynylene , lower hetroalkylene , substituted H2N H?N 65 heteroalkylene , lower heterocycloalkylen , substituted lower heterocycloalkylene , arylene , substituted arylene , het eroarylene , substituted heteroarylene , alkarylene , substi US 9 , 962, 450 B2 65 66 tuted alkarylene , aralkylene , or substituted aralkylene ; B is wherein each R , is independently selected from the group optional, and when present is a linker selected from the consisting of H , halogen , alkyl, substituted alkyl, - N ( R ') , group consisting of lower alkylene , substituted lower alky - C (O ), R ' where k is 1, 2 , or 3 , - C (O ) N (R ') 2 , - OR ', and lene , lower alkenylene , substituted lower alkenylene, lower - S (O ), R ', where each R ' is independently H , alkyl , or heteroalkylene, substituted lower heteroalkylene, - 0 , 5 substituted alkyl. - O -( alkylene or substituted alkylene ) -, - S — , - S -( alky In addition , the following amino acids are included : lene or substituted alkylene ) -, - S ( O ) k - where k is 1 , 2 , or 3 , - S ( O ) k ( alkylene or substituted alkylene )- , - C ( O ) - , - C ( O ) - ( alkylene or substituted alkylene) , C ( S ) , - C ( S ) - (alkylene or substituted alkylene ) - , - N ( R ') — , 10 - NR '- ( alkylene or substituted alkylene ) , C ( O ) N ( R ' ) — , - CON ( R ') -( alkylene or substituted alkylene )- , CSN ( R ') — , CSN ( R ') -( alkylene or substituted alkylene ) -, - N ( R ' )CO - ( alkylene or substituted alkylene ) - , — N ( R ') C 15 OH ( 0 ) O - , - S ( O ) N ( R ' ) - , - N ( R ') C ( O ) N ( R ') — , - N ( R ') C H N ( S ) N (R ') - , - N ( R ' ) S ( O ) , N ( R ') - , - N ( R ') — N — , - C (R ' ) = N — , C ( R ') = N - N ( R ') , C ( R ' ) = N N , - C (R ') 2 — N — N — , and C ( R ' )2 — N (R ') — N (R ') , where each R ' is independently H , alkyl, or substituted alkyl; 20 Rj is optional, and when present , is H , an amino protecting group , resin , amino acid , polypeptide, or polynucleotide ; and R2 is optional, and when present, is OH , an ester protecting group , resin , amino acid , polypeptide, or polynucleotide . 25 H N OH In addition , the following amino acids having the struc ture of Formula (IX ) are included :

( IX ) 30

OH H?N

35

-

2

40 B is optional, and when present is a linker selected from the QuququQiQ group consisting of lower alkylene , substituted lower alky H2N lene, lower alkenylene , substituted lower alkenylene , lower 45 heteroalkylene , substituted lower heteroalkylene , - 04 , - O - (alkylene or substituted alkylene ) - , - S — , - S - ( alky lene or substituted alkylene ) -, - S ( O ) : - where k is 1 , 2 , or 3 , - S ( O ) , ( alkylene or substituted alkylene ) - , - C ( O ) - , - C ( O ) - (alkylene or substituted alkylene ) -, - C ( S ) - , 50 - C ( S ) - ( alkylene or substituted alkylene ) -, - N ( R ') — , - NR '- ( alkylene or substituted alkylene ) -, - C ( O ) N (R ' ) — , - CON ( R ') -( alkylene or substituted alkylene ) , CSN ?? ( R ') — , CSN ( R ' ) - (alkylene or substituted alkylene ) , HN - N ( R ') CO - ( alkylene or substituted alkylene )- , - N ( R ') C 55 ( 0 ) O - , - S ( O ) , N ( R ' ) - , - N ( R ') C ( O ) N ( R ') — , - N (R ') C ( S ) N ( R ' ) — , - N ( R ') S ( O ) N ( R ') — , - N ( R ') — N — , - C (R ') — N — , - C (R ') = N / N ( R ') — , - C (R ') = N — N — , - C (R ') 2 — N — N — , and C ( R ') 2 — N (R ') — N (R ') , where each R ' is independently H , alkyl, or substituted alkyl; 60 R is H , alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R is optional, and when present, is H , an amino protecting group , resin , amino acid , polypeptide, or polynucleotide ; H2N and 65 R , is optional, and when present, is OH , an ester protecting group , resin , amino acid , polypeptide , or polynucleotide ; US 9 , 962 ,450 B2 67 68 - continued R2 is optional, and when present, is OH , an ester protecting group , resin , amino acid , polypeptide, or polynucleotide ; and each Ra is independently selected from the group consisting of H , halogen , alkyl, substituted alkyl, — N ( R ' ) 2 , - C ( O )kR ' where k is 1 , 2 , or 3 , - C ( O ) N ( R ' ) 2 , - OR ', and - S ( O )KR ', where each R ' is independently H , alkyl, or substituted alkyl; and n is 0 to 8 . HN In addition , the following amino acids are included : 10

?? OH OH HON HNH?N HONH2N ' 20 wherein such compounds are optionally amino protected , optionally carboxyl protected , optionally amino protected and carboxyl protected , or a salt thereof. In addition , these non -natural amino acids and any of the following non NH natural amino acids may be incorporated into a non -natural amino acid polypeptide . OH OH In addition , the following amino acids having the struc - 30 HONH2N H2N ture of Formula ( X ) are included :

an OH HNNYH» HNH2N 40

O , and wherein B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene , substituted lower alkalk. 45 enylene, lower heteroalkylene, substituted lower heteroalky lene , 0 , 0 - (alkylene or substituted alkylene ) . - S , - S - ( alkylene or substituted alkylene )- , - S ( O ) where k is 1 , 2 , or 3 , - S ( O ) ( alkylene or substituted 50 OH OH alkylene ) - , - C ( O ) - , - C ( O ) - ( alkylene or substituted alky H2N L HNH2N lene ) , C ( S ) , C ( S ) - ( alkylene or substituted alky lene ) - , - N ( R ' ) — , - NR ' - ( alkylene or substituted alky lene ) -, - C (O ) N ( R ') — , CON ( R ') - ( alkylene or substituted alkylene ) - , CSN ( R ') — , - CSN ( R ') - (alkylene or substi - 55 wherein such compounds are optionally amino protected , tuted alkylene) -, - N ( R ' )CO - ( alkylene or substituted alky optionally carboxyl protected , optionally amino protected lene ) -, - N ( R ') C ( O ) O - , - S ( O ) , N ( R ') - , - N ( R ') C ( O ) N and carboxyl protected , or a salt thereof. In addition , these ( R ' ) , - N ( R ' ) C ( S ) N ( R ' ) - N ( R ' ) S ( O ) N ( R ' ) — , non - natural amino acids and any of the following non — N ( R ') — N — , C ( R ') = N — , C ( R ') = N / N ( R ') — , - C ( R ') — N — N — , _ C ( R ') 2 — N — N — , and — C (R ') 2 - N 60 natural amino acids may be incorporated into a non -natural ( R ') — N ( R ') — , where each R ' is independently H , alkyl, or O amino acid polypeptide. substituted alkyl; In addition to monocarbonyl structures, the non -natural R is H , alkyl, substituted alkyl, cycloalkyl, or substituted amino acids described herein may include groups such as cycloalkyl; dicarbonyl , dicarbonyl like , masked dicarbonyl and pro R1 is optional, and when present, is H , an amino protecting 65 tected dicarbonyl groups. group , resin , amino acid , polypeptide, or polynucleotide; For example , the following amino acids having the struc and ture of Formula (XI ) are included : US 9 , 962, 450 B2 70 - C ( O )- (alkylene or substituted alkylene ) , C (S ) , ( XI) C ( S ) - ( alkylene or substituted alkylene ) , — N ( R ' ) — , - NR ' - alkylene or substituted alkylene) - , - C ( O ) N ( R ') — , CON ( R ') -( alkylene or substituted alkylene) - , CSN 5 ( R ') — , CSN ( R ') - ( alkylene or substituted alkylene ) -, ? - N ( R ') CO - ( alkylene or substituted alkylene ) -, - N ( R ') C Ri ( 0 ) O - , - S ( O ) N ( R ') — , - N ( R ') C ( O ) N ( R ') — , - N ( R ') C ( S ) N (R ') - , - N ( R ') S ( O ) , N (R ' ) — , - N ( R ') — N — , - C ( R ' ) = N — , C ( R ') = N — N ( R ') — , C ( R ') = N — N — , ' - C ( R ') 2 — N — N — , and C ( R ') 2 — N ( R ') — N ( R ') — , wherein A is optional, and when present is lower alkylene , where each R ' is independently H , alkyl, or substituted alkyl; substituted lower alkylene, lower cycloalkylene, substituted R is H , alkyl, substituted alkyl, cycloalkyl, or substituted lower cycloalkylene , lower alkenylene , substituted lower cycloalkyl; alkenylene, alkynylene , lower heteroalkylene, substituted 15 R1 is optional, and when present, is H , an amino protecting heteroalkylene , lower heterocycloalkylene , substituted group , resin , amino acid , polypeptide , or polynucleotide ; lower heterocycloalkylene, arylene, substituted arylene , het - and eroarylene , substituted heteroarylene , alkarylene , substi - R2 is optional, and when present, is OH , an ester protecting tuted alkarylene , aralkylene , or substituted aralkylene ; B is group, resin , amino acid , polypeptide, or polynucleotide; optional, and when present is a linker selected from the 20 wherein each Ra is independently selected from the group group consisting of lower alkylene, substituted lower alky - consisting of H , halogen , alkyl, substituted alkyl, — N ( R ' ) 2 , lene , lower alkenylene , substituted lower alkenylene , lower C ( O )kR ' where k is 1 , 2 , or 3 , - C ( O ) N ( R ' ) 2 , - OR ', and heteroalkylene , substituted lower heteroalkylene , 0 , - S ( O )kR ', where each R ' is independently H , alkyl, or 0 - ( alkylene or substituted alkylene )- , - S , S - ( alkylene or substituted alkyl. substituted alkylene ) - , - S ( O ) k - where k is 1 , 2 , or 3 , 25 In addition , the following amino acids are included : - S ( O ) k ( alkylene or substituted alkylene) - , C (O ) , C ( O ) ( alkylene or substituted alkylene ) -, - C ( S ) , C ( S ) - ( alky lene or substituted alkylene ) - , - N ( R ') — , NR ' - ( alkylene or substituted alkylene )- , C ( O ) N ( R ') — , CON ( R ') - (alkylene or substituted alkylene ) -, CSN ( R ') — , CSN ( R ') - ( alkylene or 30 and substituted alkylene ) - , N ( R ' ) CO - (alkylene or substituted alkylene ) , N ( R ') C ( O ) O - , S ( O ) , N ( R ') — , N ( R ') C ( O ) N ( R ') — , N ( R ') C ( S ) N ( R ') — , N ( R ') S ( O )KN ( R ') — , N ( R ') — N = , - C ( R ' ) = N — , C ( R ') = N - N ( R ' ) , C ( R ' ) — N — N — , C ( R ' ) 2 - N — N — and C ( R ') 2N ( R ') N ( R ') , 35 H?N COOH where each R ' is independently H , alkyl, or substituted alkyl; R is H , alkyl , substituted alkyl, cycloalkyl , or substituted cycloalkyl; R1 is optional, and when present, is H , an amino protecting group , resin , amino acid , polypeptide, or polynucleotide ; 40 and R2 is optional, and when present, is OH , an ester protecting group , resin , amino acid , polypeptide , or polynucleotide . H?N COOH In addition , the following amino acids having the struc ture of Formula (XII ) are included : 45 wherein such compounds are optionally amino protected , optionally carboxyl protected , optionally amino protected and carboxyl protected , or a salt thereof. In addition , these (XII ) non - natural amino acids and any of the following non natural amino acids may be incorporated into a non -natural 50 amino acid polypeptide . In addition , the following amino acids having the struc ture of Formula (XIII ) are included :

- 55 XIII ) N (CRang

60 B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alky N lene, lower alkenylene , substituted lower alkenylene , lower heteroalkylene, substituted lower heteroalkylene , - 04 , - O -( alkylene or substituted alkylene ) -, - S — , - S - alky - 65 wherein B is optional, and when present is a linker selected lene or substituted alkylene ) - , - S ( O ) - where k is 1 , 2 , or from the group consisting of lower alkylene , substituted 3 , S ( O ) k (alkylene or substituted alkylene ) -, - C ( O ) - , lower alkylene , lower alkenylene , substituted lower alk US 9 ,962 , 450 B2 71 72 enylene, lower heteroalkylene, substituted lower heteroalky - continued lene, 0 , 0 - (alkylene or substituted alkylene )- , S , S -( alkylene or substituted alkylene ) -, S ( 0 ); where k is 1 , 2 , or 3 , S ( 0 ); ( alkylene or substituted alkylene) -, - C ( 0 ) , C ( 0 ) -( alkylene or substituted alky lene) - , - C ( S ) , C ( S )- (alkylene or substituted alky NH lene ) -, - N ( R ' ) , NR '- ( alkylene or substituted alky lene ) -, - C ( 0 ) N ( R ') , CON ( R ') -( alkylene or substituted 0H alkylene )- , CSN ( R ' ) , CSN ( R ' ) -( alkylene or substi - 10 H5N tuted alkylene ) -, NR' ) CO - ( alkylene or substituted alky H2N lene ) -, NR' ) C ( O ) 0 , S ( 0 ) N ( R ' ) , N ( R ' ) C ( O ) N ( R ') , NR' ) C ( S ) N ( R ') , N ( R ') S ( O ) N (R ') , 0 N ( R ') N , C ( R ' ) N , C ( R ') N N ( R ') , 15 CR' ) NN , C ( R ') NN , and C ( R ') N ( R ') N ( R ') , where each R ' is independently H , alkyl, or substituted alkyl ; R is H , alkyl, substituted alkyl , cycloalkyl , or substituted ?? cycloalkyl; Rj is optional, and when present , is H , an amino protecting 0F group , resin , amino acid , polypeptide , or polynucleotide ; HN HN and R , is optional, and when present , is OH , an ester protecting 25 group , resin , amino acid , polypeptide , or polynucleotide; each R , is independently selected from the group consist 0??? ing of H , halogen , alkyl, substituted alkyl, NR' ) , C ( 0 ) R ' where kis 1 , 2 , or 3 , - CON ( R ' ) , OR ', and 30 NH S ( 0 ) R ', where each R is independently H , alkyl , or substituted alkyl ; and nis 0 to 8 . In addition , the following amino acids are included : ? OH 0H 35 ? H2N HaN 0 0 0

0H 0H HN HoN

0H HoN H5N

NH 0

10H 0H H5N HN HNO , and

66) ? OH .0H H5N H5N

CH .0H H5N HN * 65 wherein such compounds are optionally amino protected , optionally carboxyl protected , optionally amino protected and carboxyl protected , or ????a salt thereof. In addition , these US 9 ,962 ,450 B2 73 74 non -natural amino acids and any of the following non In addition , the following amino acids having the struc natural amino acids may be incorporated into a non -natural ture of Formula ( XIV - B ) are included : amino acid polypeptide. In addition , the following amino acids having the struc ture of Formula (XIV ) are included : 5 XIV - B ) 0 0 ( XIV ) OE 1 R 10 R ; R ,HN C ( O )RZ

RHN C( O) R2 wherein : 15 A is optional , and when present is lower alkylene , substi wherein : tuted lower alkylene , lower cycloalkylene , substituted lower A is optional , and when present is lower alkylene, substi cycloalkylene , lower alkenylene , substituted lower alk tuted lower alkylene, lower cycloalkylene, substituted lower enylene, alkynylene , lower heteroalkylene , substituted het cycloalkylene , lower alkenylene , substituted lower alk eroalkylene , lower heterocycloalkylene , substituted lower enylene, alkynylene , lower heteroalkylene , substituted het - 20 heterocycloalkylene, arylene , substituted arylene , het eroalkylene , lower heterocycloalkylene , substituted lower eroarylene , substituted heteroarylene, alkarylene , substi heterocycloalkylene , arylene , substituted arylene , het - tuted alkarylene , aralkylene , or substituted aralkylene ; eroarylene , substituted heteroarylene , alkarylene , substi- Ris H , alkyl, substituted alkyl, cycloalkyl, or substituted tuted alkarylene , aralkylene , or substituted aralkylene ; cycloalkyl; R is H , alkyl , substituted alkyl, cycloalkyl , or substituted 25 R1 is optional, and when present, is H , an amino protecting cycloalkyl; group , resin , amino acid , polypeptide , or polynucleotide ; R1 is optional, and when present, is H , an amino protecting and group , resin , amino acid , polypeptide , or polynucleotide ; R2 is optional, and when present, is OH , an ester protecting and group , resin , amino acid , polypeptide , or polynucleotide ; R2 is optional, and when present, is OH , an ester protecting 30 L is alkylene, substituted alkylene , N (R ') ( alkylene ) or N (R ') group , resin , amino acid , polypeptide, or polynucleotide; (substituted alkylene ) , where R ' is H , alkyl, substituted alkyl , X1 is C , S , or S ( O ) ; and L is alkylene , substituted alkylene , cycloalkyl, or substituted cycloalkyl. N ( R ') ( alkylene ) or N ( R ') ( substituted alkylene ), where R ' is In addition , the following amino acids having the struc H , alkyl, substituted alkyl, cycloalkyl, or substituted ture of Formula (XV ) are included : cycloalkyl. 3535 In addition , the following amino acids having the struc ture of Formula (XIV - A ) are included : 0 (XV ) (XIV - A ) 40 A ( CRSR %) n R ;

R RHN ( O ) R2 45 wherein : RAHN C ( O )R2 A is optional, and when present is lower alkylene, substi tuted lower alkylene, lower cycloalkylene , substituted lower wherein : cycloalkylene , lower alkenylene , substituted lower alk A is optional, and when present is lower alkylene, substi - 50 enylene, alkynylene , lower heteroalkylene , substituted het tuted lower alkylene , lower cycloalkylene , substituted lower eroalkylene , lower heterocycloalkylene , substituted lower cycloalkylene , lower alkenylene , substituted lower alk - heterocycloalkylene , arylene , substituted arylene, het enylene , alkynylene , lower heteroalkylene , substituted het eroarylene , substituted heteroarylene , alkarylene, substi eroalkylene , lower heterocycloalkylene , substituted lower tuted alkarylene, aralkylene , or substituted aralkylene ; heterocycloalkylene, arylene , substituted arylene , het - 55 R is H , alkyl, substituted alkyl, cycloalkyl, or substituted eroarylene , substituted heteroarylene , alkarylene, substi cycloalkyl ; tuted alkarylene , aralkylene , or substituted aralkylene ; R , is optional, and when present, is H , an amino protecting R is H , alkyl, substituted alkyl, cycloalkyl, or substituted group , resin , amino acid , polypeptide, or polynucleotide ; cycloalkyl; and R1 is optional, and when present, is H , an amino protecting 60 R , is optional, and when present, is OH , an ester protecting group , resin , amino acid , polypeptide , or polynucleotide ; group , resin , amino acid , polypeptide, or polynucleotide ; and X , is C , S , or S ( O ) ; and n is 0 , 1 , 2 , 3 , 4 , or 5 ; and each R $ R2 is optional, and when present, is OH , an ester protecting and R on each CR?R group is independently selected from group , resin , amino acid , polypeptide , or polynucleotide ; the group consisting of H , alkoxy, alkylamine , halogen , L is alkylene , substituted alkylene , N ( R ') ( alkylene ) or N ( R ' ) 65 alkyl, aryl, or any R $ and Rº can together form = O or a ( substituted alkylene ) , where R ' is H , alkyl, substituted alkyl, cycloalkyl , or any to adjacent R $ groups can together form cycloalkyl , or substituted cycloalkyl . a cycloalkyl . US 9 , 962 , 450 B2 75 76 In addition , the following amino acids having the struc can together form = 0 or a cycloalkyl, or any to adjacent R $ ture of Formula (XV - A ) are included : groups can together form a cycloalkyl . In addition , the following amino acids having the struc ture of Formula ( XVI) are included : (XV - A ) 5

(XVI )

A ( CRR ) R O=> 10 AL R ; RHN CC( O) R , wherein : RHN C( O ) R2 A is optional , and when present is lower alkylene , substi - 15 tuted lower alkylene , lower cycloalkylene, substituted lower wherein : cycloalkylene , lower alkenylene , substituted lower alk A is optional , and when present is lower alkylene , substi enylene, alkynylene , lower heteroalkylene , substituted het - tuted lower alkylene , lower cycloalkylene , substituted lower eroalkylene , lower heterocycloalkylene , substituted lower cycloalkylene , lower alkenylene , substituted lower alk heterocycloalkylene , arylene , substituted arylene, het - 20 enylene, alkynylene , lower heteroalkylene , substituted het eroarylene, substituted heteroarylene , alkarylene , substi eroalkylene , lower heterocycloalkylene , substituted lower tuted alkarylene, aralkylene , or substituted aralkylene ; heterocycloalkylene , arylene, substituted arylene , het R is H , alkyl, substituted alkyl, cycloalkyl, or substituted eroarylene , substituted heteroarylene , alkarylene , substi cycloalkyl; tuted alkarylene , aralkylene , or substituted aralkylene ; R , is optional , and when present, is H , an amino protecting 25 R is H , alkyl, substituted alkyl, cycloalkyl, or substituted group , resin , amino acid , polypeptide , or polynucleotide; cycloalkyl; and Rj is optional , and when present, is H , an amino protecting R , is optional , and when present, is OH , an ester protecting group , resin , amino acid , polypeptide , or polynucleotide; and group , resin , amino acid , polypeptide , or polynucleotide ; 9 30 R2 is optional, and when present, is OH , an ester protecting n is 0 , 1 , 2 , 3 , 4 , or 5 ; and each R8 and R on each CRØR group , resin , amino acid , polypeptide, or polynucleotide ; group is independently selected from the group consisting of X , is C , S , or S ( O ) ; and L is alkylene , substituted alkylene , H , alkoxy, alkylamine , halogen , alkyl, aryl, or any R $ and R N ( R ' ) (alkylene ) or N ( R ') ( substituted alkylene ) , where R ' is can together form = 0 or a cycloalkyl , or any to adjacent R $ H , alkyl , substituted alkyl , cycloalkyl, or substituted groups can together form a cycloalkyl. 35 cycloalkyl. In addition , the following amino acids having the struc In addition , the following amino acids having the struc ture of Formula (XV - B ) are included : ture of Formula (XVI - A ) are included :

(XV - B ) 40 (XVI - A )

(CRR ) R N — L R

45 RHN ( c( O) R , RHNRAHN C (O )R2 wherein : wherein : A is optional, and when present is lower alkylene, substi- 50 A is optional , and when present is lower alkylene , substi tuted lower alkylene, lower cycloalkylene, substituted lower tuted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene , lower alkenylene , substituted lower alk - cycloalkylene , lower alkenylene , substituted lower alk enylene , alkynylene , lower heteroalkylene , substituted het enylene , alkynylene , lower heteroalkylene , substituted het eroalkylene, lower heterocycloalkylene, substituted lower eroalkylene, lower heterocycloalkylene , substituted lower heterocycloalkylene, arylene , substituted arylene , het - 55 heterocycloalkylene , arylene , substituted arylene , het eroarylene , substituted heteroarylene , alkarylene , substi- eroarylene, substituted heteroarylene , alkarylene , substi tuted alkarylene , aralkylene , or substituted aralkylene ; tuted alkarylene , aralkylene , or substituted aralkylene ; R is H , alkyl, substituted alkyl, cycloalkyl, or substituted R is H , alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; cycloalkyl; R is optional , and when present, is H , an amino protecting 60 R is optional , and when present, is H , an amino protecting group , resin , amino acid , polypeptide, or polynucleotide ; group , resin , amino acid , polypeptide , or polynucleotide ; and and R , is optional, and when present, is OH , an ester protecting R , is optional , and when present, is OH , an ester protecting group , resin , amino acid , polypeptide , or polynucleotide ; group , resin , amino acid , polypeptide, or polynucleotide ; n is 0 , 1 , 2 , 3 , 4 , or 5 ; and each RP and Rº on each CRØR 65 L is alkylene , substituted alkylene , N ( R ') ( alkylene) or N ( R ') group is independently selected from the group consisting of ( substituted alkylene ) , where R ' is H , alkyl, substituted alkyl, H , alkoxy, alkylamine, halogen , alkyl, aryl , or any R $ andR c ycloalkyl, or substituted cycloalkyl . US 9 ,962 , 450 B2 77 78 In addition , the following amino acids having the struc - continued ture of Formula (XVI - B ) are included : ( b ) (b ) man

n (XVI - B ) 5 2 S aims PTT(a ) 5 ( 6 ) ( b ) N - 1 R m I man

10

R?HN C (O )R2 w DI(a ) ( b ) a wherein : A is optional, and when present is lower alkylene , substi - 15 mm tuted lower alkylene, lower cycloalkylene, substituted lower mo nin cycloalkylene , lower alkenylene , substituted lower alk 4( a ) . ( a ) enylene , alkynylene , lower heteroalkylene , substituted het eroalkylene , lower heterocycloalkylene , substituted lower heterocycloalkylene, arylene , substituted arylene, het - 20 where (a ) indicates bonding to the A group and (b ) indicates eroarylene, substituted heteroarylene, alkarylene, substi bonding to respective carbonyl groups , R , and R . are inde pendently chosen from H , halogen , alkyl, substituted alkyl, tuted alkarylene, aralkylene , or substituted aralkylene ; cycloalkyl, or substituted cycloalkyl, or Rz and R4 or two Rz R is H , alkyl , substituted alkyl, cycloalkyl, or substituted groups or two R4 groups optionally form a cycloalkyl or a cycloalkyl; heterocycloalkyl; R is optional, and when present, is H , an amino protecting R is H , halogen , alkyl, substituted alkyl, cycloalkyl , or group , resin , amino acid , polypeptide , or polynucleotide ; substituted cycloalkyl ; and Tz is a bond , C ( R ) ( R ) , O , or S , and R is H , halogen , alkyl, R , is optional, and when present, is OH , an ester protecting substituted alkyl, cycloalkyl, or substituted cycloalkyl; group , resin , amino acid , polypeptide , or polynucleotide ; 30 R is optional , and when present , is H , an amino protecting L is alkylene , substituted alkylene , N (R ') ( alkylene ) or N (R ') group , resin , amino acid , polypeptide, or polynucleotide ; ( substituted alkylene ), where R ' is H , alkyl, substituted alkyl, and cycloalkyl, or substituted cycloalkyl. R2 is optional, and when present, is OH , an ester protecting In addition , amino acids having the structure of Formula group , resin , amino acid , polypeptide, or polynucleotide. (XVII ) are included : In addition , amino acids having the structure of Formula (XVIII ) are included : (XVII ) R . (XVIII ) RO R3 M R3LA 13 Ra R NH -

IZ wherein : A is optional, and when present is lower alkylene, substi - > tuted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene , lower alkenylene, substituted lower alk wherein : enylene, alkynylene , lower heteroalkylene , substituted het - Mis C ( R3) — , eroalkylene , lower heterocycloalkylene , substituted lower heterocycloalkylene, arylene , substituted arylene, het - » eroarylene , substituted heteroarylene , alkarylene , substi ( b ) tuted alkarylene , aralkylene , or substituted aralkylene ; w ma Mis - C (R3 ) , 60 nen RA RAS , (a mm ) ( b ) (6 ) mann namun

mvh2) R4 R43 , 101 R43 , 65 menn w ma US 9, 962 ,450 B2 79 - continued ( b ) ( XX ) ma ma

www (Q ) (a )

10 (8 )

w n

un 2 or mins sms , or wb( a ) 15 wherein : R is H , halogen , alkyl , substituted alkyl, cycloalkyl, or substituted cycloalkyl . In addition , the following amino acids having structures of Formula ( XXI) are included : where (a ) indicates bonding to the A group and (b ) indicates 20 bonding to respective carbonyl groups, Rz and R4 are inde pendently chosen from H , halogen , alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, or Rz and R4 or two Rz groups or two R4 groups optionally form a cycloalkyl or a 25 O , andand heterocycloalkyl ; R is H , halogen , alkyl , substituted alkyl , cycloalkyl, or substituted cycloalkyl ; Tz is a bond, C ( R ) R ), O , or S , and R is H , halogen , alkyl, 30 substituted alkyl, cycloalkyl, or substituted cycloalkyl; N R is optional, and when present , is H , an amino protecting group , resin , amino acid , polypeptide , or polynucleotide ; and R , is optional , and when present, is OH , an ester protecting 35 group , resin , amino acid , polypeptide , or polynucleotide; each R , is independently selected from the group consisting of H , halogen , alkyl, substituted alkyl, — N ( R ') 2, - C ( O ) R ' where k is 1 , 2 , or 3 , C (O ) N (R ') 2 , — OR ', and - S ( O ), R ', 40 where each R ' is independently H , alkyl, or substituted alkyl. In addition , amino acids having the structure of Formula (XIX ) are included : 45 In some embodiments , a polypeptide comprising a non (XIX ) natural amino acid is chemically modified to generate a R 0 reactive carbonyl or dicarbonyl functional group . For instance , an aldehyde functionality useful for conjugation 50 reactions can be generated from a functionality having adjacent amino and hydroxyl groups . Where thebiologically 12 active molecule is a polypeptide , for example , an N - terminal serine or threonine ( which may be normally present or may be exposed via chemical or enzymatic digestion ) can be used 55 to generate an aldehyde functionality under mild oxidative EZ cleavage conditions using periodate . See , e . g ., Gaertner, et . al. , Bioconjug. Chem . 3 : 262 - 268 ( 1992 ) ; Geoghegan , K . & Stroh , J ., Bioconjug . Chem . 3 : 138 - 146 (1992 ) ; Gaertner et al. , J . Biol. Chem . 269 :7224 - 7230 ( 1994 ) . However, meth 60 ods known in the art are restricted to the amino acid at the wherein : N - terminus of the peptide or protein . In the present invention , a non -natural amino acid bearing R is H , halogen , alkyl, substituted alkyl, cycloalkyl, or adjacent hydroxyl and amino groups can be incorporated substituted cycloalkyl; and into the polypeptide as a " masked ” aldehyde functionality. Tz is O , or S . 65 For example , 5 -hydroxylysine bears a hydroxyl group adja In addition , amino acids having the structure of Formula cent to the epsilon amine. Reaction conditions for generating (XX ) are included : the aldehyde typically involve addition of molar excess of US 9 , 962, 450 B2 82 sodium metaperiodate under mild conditions to avoid oxi Glutamine analogues as Potential Antimalarials , Eur. J. Med . dation at other sites within the polypeptide . The pH of the Chem . 26 , 201- 5 ; Koskinen , A . M . P . & Rapoport, H . ( 1989 ) oxidation reaction is typically about 7 .0 . A typical reaction Synthesis of 4 - Substituted Prolines as Conformationally involves the addition of about 1 . 5 molar excess of sodium Constrained Amino Acid Analogues . J . Org . Chem . 54 , meta periodate to a buffered solution of the polypeptide , 5 1859 - 1866 ; Christie , B . D . & Rapoport , H . ( 1985 ) Synthesis followed by incubation for about 10 minutes in the dark . of Optically Pure Pipecolates from L - Asparagine . Applica See , e . g . U . S . Pat. No . 6 , 423 ,685 . tion to the Total Synthesis of ( + )- Apovincamine through The carbonyl or dicarbonyl functionality can be reacted Amino Acid Decarbonylation and Iminium Ion Cyclization . selectively with a hydroxylamine -containing reagent under J . Org . Chem . 50 : 1239 - 1246 ; Barton et al. , ( 1987 ) Synthesis mild conditions in aqueous solution to form the correspond - 10 of Novel alpha -Amino - Acids and Derivatives Using Radical ing oxime linkage that is stable under physiological condi Chemistry : Synthesis of L - and D - alpha - Amino -Adipic tions . See , e. g. , Jencks, W . P. , J. Am . Chem . Soc. 81 , Acids, L - alpha -aminopimelic Acid and Appropriate Unsatu 475 - 481 ( 1959 ) ; Shao , J . and Tam , J . P . , J . Am . Chem . Soc . rated Derivatives . Tetrahedron 43 :4297 - 4308 ; and , Subas 117 : 3893 - 3899 ( 1995 ) . Moreover, the unique reactivity of inghe et al ., ( 1992 ) Quisqualic acid analogues : synthesis of fication in the presence of the other amino acid side chains . beta -heterocyclic 2 -aminopropanoic acid derivatives and See , e . g . , Cornish , V . W ., et al. , J . Am . Chem . Soc . 118 : their activity at a novel quisqualate - sensitized site . J. Med . 8150 -8151 ( 1996 ); Geoghegan , K . F . & Stroh , J. G . , Bio Chem . 35 : 4602 - 7 . See also , U . S . Patent Publication No. US conjug . Chem . 3 : 138 - 146 ( 1992 ) ; Mahal. L . K ., et al. , 2004 /0198637 entitled “ Protein Arrays , ” which is incorpo Science 276 : 1125 - 1128 ( 1997 ) . 20 rated by reference herein . Structure and Synthesis of Non -Natural Amino Acids : A . Carbonyl Reactive Groups Hydroxylamine -Containing Amino Acids Amino acids with a carbonyl reactive group allow for a U .S . patent application Ser. No . 11/ 316 ,534 (U . S . Publi- variety of reactions to link molecules ( including but not cation No . 20060189529 ) is incorporated by reference in its limited to , PEG or other water soluble molecules ) via entirety . Thus , the disclosures provided in Section V ( en - 25 nucleophilic addition or aldol condensation reactions among titled “ Non -natural Amino Acids ” ), Part B ( entitled “ Struc - others. ture and Synthesis of Non -Natural Amino Acids: Hydrox - Exemplary carbonyl- containing amino acids can be rep ylamine - Containing Amino Acids " ) , in U . S . patent resented as follows: application Ser . No . 11 /316 , 534 ( U . S . Publication No . 20060189529 ) apply fully to the methods, compositions 30 ( including Formulas I -XXXV ) , techniques and strategies for (CH2 ) ?RICOR2 making, purifying , characterizing, and using non -natural amino acids, non -natural amino acid polypeptides and modi fied non -natural amino acid polypeptides described herein to R3HN CORA the same extent as if such disclosures were fully presented 35 herein . U . S . Patent Publication Nos . 2006 /0194256 , 2006 / wherein n is 0 - 10 ; R , is an alkyl, aryl, substituted alkyl, or 0217532 , and 2006 /0217289 and WO 2006 /069246 entitled substituted aryl; R , is H , alkyl, aryl, substituted alkyl, and “ Compositions containing, methods involving , and uses of substituted aryl ; and R3 is H , an amino acid , a polypeptide , non -natural amino acids and polypeptides, ” are also incor or an amino terminus modification group , and R4 is H , an porated herein by reference in their entirety . 40 amino acid , a polypeptide , or a carboxy terminus modifica Chemical Synthesis of Unnatural Amino Acids tion group . In some embodiments , n is 1 , R , is phenyl and Many of the unnatural amino acids suitable for use in the R , is a simple alkyl ( i. e ., methyl , ethyl, or propyl) and the present invention are commercially available , e . g . , from ketone moiety is positioned in the para position relative to Sigma (USA ) or Aldrich (Milwaukee , Wis . , USA ) . Those the alkyl side chain . In some embodiments , n is 1 , Rj is that are not commercially available are optionally synthe - 45 phenyl and R , is a simple alkyl ( i. e ., methyl, ethyl, or sized as provided herein or as provided in various publica - propyl) and the ketone moiety is positioned in the meta tions or using standard methods known to those of ordinary position relative to the alkyl side chain . skill in the art . For organic synthesis techniques, see , e . g ., The synthesis of p -acetyl - ( + / - ) -phenylalanine and Organic Chemistry by Fessendon and Fessendon , ( 1982 , m -acetyl - ( + - ) - phenylalanine is described in Zhang , Z ., et Second Edition , Willard Grant Press , Boston Mass. ) ; 50 al. , Biochemistry 42 : 6735 -6746 ( 2003 ) , which is incorpo Advanced Organic Chemistry by March ( Third Edition , rated by reference herein . Other carbonyl - containing amino 1985 , Wiley and Sons, New York ) ; and Advanced Organic acids can be similarly prepared by one of ordinary skill in Chemistry by Carey and Sundberg ( Third Edition , Parts A the art . and B , 1990 , Plenum Press , New York ) . Additional publi - In some embodiments , a polypeptide comprising a non cations describing the synthesis of unnatural amino acids 55 naturally encoded amino acid is chemically modified to include, e .g ., WO 2002 /085923 entitled “ In vivo incorpora generate a reactive carbonyl functional group . For instance , tion of Unnatural Amino Acids ;” Matsoukas et al. , ( 1995 ) J . an aldehyde functionality useful for conjugation reactions Med . Chem . , 38 , 4660 -4669 ; King, F . E . & Kidd , D . A . A . can be generated from a functionality having adjacent amino ( 1949) A New Synthesis of Glutamine and of y -Dipeptides and hydroxyl groups . Where the biologically active mol of Glutamic Acid from Phthylated Intermediates. J. Chem . 60 ecule is a polypeptide , for example , an N - terminal serine or Soc. , 3315 - 3319 ; Friedman , O . M . & Chattenji, R . ( 1959 ) threonine (which may be normally present or may be Synthesis of Derivatives of Glutamine as Model Substrates exposed via chemical or enzymatic digestion ) can be used to DA for Anti - Tumor Agents . J . Am . Chem . Soc . 81, 3750 -3752 2 ;, generate an aldehyde functionality under mild oxidative Craig, J. C . et al. (1988 ) Absolute Configuration of the cleavage conditions using periodate . See , e . g . , Gaertner, et Enantiomers of 7 -Chloro - 4 [ 4 - ( diethylamino ) - 1 -methylbu - 65 al ., Bioconjug . Chem . 3 : 262 - 268 ( 1992 ) ; Geoghegan , K . & tyl?amino ] quinoline (Chloroquine ) . J . Org . Chem . 53 , 1167 - Stroh , J ., Bioconjug. Chem . 3 : 138 - 146 ( 1992 ) ; Gaertner et 1170 ; Azoulay , M . , Vilmont, M . & Frappier , F . ( 1991) al. , J . Biol. Chem . 269: 7224 - 7230 (1994 ). However, meth US 9 , 962, 450 B2 83 84 ods known in the art are restricted to the amino acid at the Shao , J. and Tam , J. , J Am . Chem . Soc. 117 : 3893 -3899 N -terminus of the peptide or protein . ( 1995 ). The unique reactivity of hydrazide, hydrazine and In the present invention , a non -naturally encoded amino semicarbazide functional groups makes them significantly acid bearing adjacent hydroxyl and amino groups can be more reactive toward aldehydes, ketones and other electro incorporated into the polypeptide as a “ masked ” aldehyde 5 philic groups as compared to the nucleophilic groups present functionality . For example , 5 -hydroxylysine bears a on the 20 common amino acids ( including but not limited to , hydroxyl group adjacent to the epsilon amine . Reaction the hydroxyl group of serine or threonine or the amino conditions for generating the aldehyde typically involve groups of lysine and the N - terminus) . addition of molar excess of sodium metaperiodate under C . Aminooxy - Containing Amino Acids mild conditions to avoid oxidation at other sites within the 10 Non - naturally encoded amino acids containing an ami polypeptide . The pH of the oxidation reaction is typically nooxy (also called a hydroxylamine ) group allow for reac about 7 .0 . A typical reaction involves the addition of about tion with a variety of electrophilic groups to form conjugates 1 . 5 molar excess of sodium meta periodate to a buffered ( including but not limited to , with PEG or other water solution of the polypeptide, followed by incubation for soluble polymers ). Like hydrazines, hydrazides and semi about 10 minutes in the dark . See , e . g . U . S . Pat . No . 15 6 ,423 ,685 , which is incorporated by reference herein . carbazides , the enhanced nucleophilicity of the aminooxy The carbonyl functionality can be reacted selectively with group permits it to react efficiently and selectively with a a hydrazine -, hydrazide - , hydroxylamine - , or semicarbaz variety of molecules that contain aldehydes or other func ide -containing reagent under mild conditions in aqueous tional groups with similar chemical reactivity . See , e . g . , solution to form the corresponding hydrazone oxime or 20 Shao , J . and Tam , J ., J. Am . Chem . Soc. 117 : 3893 - 3899 semicarbazone linkages, respectively , that are stable under (1995 ) ; H . Hang and C . Bertozzi , Acc. Chem . Res. 34 : physiological conditions. See , e .g ., Jencks , W . P ., J. Am . 727 - 736 (2001 ) . Whereas the result of reaction with a Chem . Soc . 81, 475 -481 ( 1959 ) ; Shao , J . and Tam , J . P ., J . hydrazine group is the corresponding hydrazone, however , Am . Chem . Soc. 117 : 3893 - 3899 ( 1995 ) . Moreover , the an oxime results generally from the reaction of an aminooxy unique reactivity of the carbonyl group allows for selective 25 group with a carbonyl- containing group such as a ketone . modification in the presence of the other amino acid side Exemplary amino acids containing aminooxy groups can chains. See, e . g . , Cornish , V . W ., et al ., J . Am . Chem . Soc. be represented as follows : 118 : 8150 -8151 ( 1996 ) ; Geoghegan , K . F. & Stroh , J . G ., Bioconjug . Chem . 3 : 138 -146 ( 1992 ) ; Mahal, L . K . , et al. , Science 276 :1125 - 1128 (1997 ) . 30 (CH2 ) , R , — X — (CH2 ) m - Y - O - NH2 B . Hydrazine , Hydrazide or Semicarbazide Reactive Groups Non -naturally encoded amino acids containing a nucleo R2HN COR3 philic group , such as a hydrazine, hydrazide or semicarba zide, allow for reaction with a variety of electrophilic groups to form coniugates (including but not limited to , with PEG 35 wherein n is 0 - 10 ; R , is an alkyl, aryl, substituted alkyl, or or other water soluble polymers ) . substituted aryl or not present ; X is O , N , S or not present; Exemplary hydrazine, hydrazide or semicarbazide- con m is 0 - 10 ; Y = C ( O ) or not present; R , is H , an amino acid , taining amino acids can be represented as follows: a polypeptide , or an amino terminus modification group , and Rz is H , an amino acid , a polypeptide, or a carboxy terminus 40 modification group . In some embodiments , n is 1 , R1 is ( CH2) , RX = C( O ) - NH - HN ) phenyl, X is O , m is 1 , and Y is present . In some embodi ments , n is 2 , R , and X are not present, m is 0 , and Y is not R2HN COR present. 45 Aminooxy - containing amino acids can be prepared from wherein n is 0 - 10 ; R , is an alkyl, aryl, substituted alkyl, or readily available amino acid precursors (homoserine , serine substituted aryl or not present ; X , is O , N , or S or not and threonine ). See , e . g . , M . Carrasco and R . Brown, J. Org. present; R , is H , an amino acid , a polypeptide , or an amino Chem . 68 : 8853 - 8858 (2003 ) . Certain aminooxy - containing terminus modification group , and Rz is H , an amino acid , a amino acids, such as L -2 -amino - 4 -( aminooxy )butyric acid ), polypeptide , or a carboxy terminus modification group . 50 have been isolated from natural sources (Rosenthal , G ., Life In some embodiments , n is 4 , R , is not present, and X is Sci. 60 : 1635 - 1641 (1997 ). Other aminooxy -containing N . In some embodiments , n is 2 , R , is not present , and X is amino acids can be prepared by one of ordinary skill in the not present. In some embodiments , n is 1 , R , is phenyl, X is art . O , and the oxygen atom is positioned para to the aliphatic D . Azide and Alkyne Reactive Groups group on the aryl ring . 55 The unique reactivity of azide and alkyne functional Hydrazide- , hydrazine - , and semicarbazide- containing groups makes them extremely useful for the selective modi amino acids are available from commercial sources. For fication of polypeptides and other biological molecules . instance , L - glutamate - O -hydrazide is available from Sigma Organic azides , particularly aliphatic azides, and alkynes are Chemical (St . Louis , Mo. ) . Other amino acids not available generally stable toward common reactive chemical condi commercially can be prepared by one of ordinary skill in the 60 tions. In particular , both the azide and the alkyne functional art . See , e . g ., U . S . Pat. No. 6 , 281, 211 , which is incorporated groups are inert toward the side chains ( i . e . , R groups ) of the by reference herein . 20 common amino acids found in naturally -occurring poly Polypeptides containing non -naturally encoded amino peptides . When brought into close proximity , however , the acids that bear hydrazide , hydrazine or semicarbazide func - " spring - loaded ” nature of the azide and alkyne groups is tionalities can be reacted efficiently and selectively with a 65 revealed and they react selectively and efficiently via Huis variety of molecules that contain aldehydes or other func - gen [ 3 + 2 ] cycloaddition reaction to generate the correspond tional groups with similar chemical reactivity . See , e .g ., ing triazole . See , e .g . , Chin J ., et al ., Science 301: 964 - 7 US 9 , 962 , 450 B2 85 86 ( 2003 ) ; Wang , Q ., et al. , J . Am . Chem . Soc. 125 , 3192 - 3193 each of the R groups is independently selected as are each ( 2003 ); Chin , J. W ., et al. , J. Am . Chem . Soc. 124 :9026 -9027 R ', R " , R '' and R " " groups when more than one of these ( 2002) . groups is present. When R ' and R " are attached to the same Because the Huisgen cycloaddition reaction involves a nitrogen atom , they can be combined with the nitrogen atom selective cycloaddition reaction (see , e . g . , Padwa , A ., in 5 to form a 5 - , 6 -, or 7 -membered ring . For example , - NR ' R " COMPREHENSIVE ORGANIC SYNTHESIS, Vol. 4 , ( ed . Trost , B . M ., is meant to include, but not be limited to , 1 - pyrrolidinyl and 1991 ) , p . 1069 - 1109 ; Huisgen , R . in 1, 3 - DIPOLAR CYCLOAD - 4 -morpholinyl . From the above discussion of substituents , DITION CHEMISTRY, (ed . Padwa, A ., 1984 ), p . 1 - 176 ) rather than one of skill in the art will understand that the term “ alkyl” a nucleophilic substitution , the incorporation of non -natu is meant to include groups including carbon atoms bound to rally encoded amino acids bearing azide and alkyne - con - " groups other than hydrogen groups , such as haloalkyl ( in taining side chains permits the resultant polypeptides to be cluding but not limited to , — CF , and CH CF3 ) and acyl modified selectively at the position of the non - naturally ( including but not limited to , - C ( O ) CHz, - C ( O )CF3 , encoded amino acid . Cycloaddition reaction involving azide C (O )CH OCHz, and the like ). or alkyne - containing relaxin polypeptide can be carried out The azide functional group can also be reacted selectively at room temperature under aqueous conditions by the addi- 1. with a water soluble polymer containing a thioester and tion of Cu ( II) ( including but not limited to , in the form of a appropriately functionalized with an aryl phosphine moiety catalytic amount of CuSO4) in the presence of a reducing agent for reducing Cu ( II) to Cu ( I ) , in situ , in catalytic to generate an amide linkage . The aryl phosphine group amount. See , e . g . , Wang, Q . , et al. , J. Am . Chem . Soc . 125 , reduces the azide in situ and the resulting amine then reacts 3192 -3193 ( 2003 ) ; Tornoe , C . W ., et al. , J . Org . Chem ?.? 220 efficiently with the thioester linkage to generate the corre 67 : 3057 - 3064 (2002 ) ; Rostovtsev, et al. , Angew . Chem . Int. sponding amide. Exemplary water soluble polymers con Ed 41: 2596 -2599 (2002 ). Exemplary reducing agents taining a thioester and a phosphine moiety can be repre include, including but not limited to , ascorbate , metallic sented as follows: copper , quinine , hydroquinone , vitamin K , glutathione , cys teine, Fe2 +, Co2 + , and an applied electric potential. 25 In some cases , where a Huisgen [ 3 + 2 ] cycloaddition Ph) P ( HC), reaction between an azide and an alkyne is desired , the relaxin polypeptide comprises a non - naturally encoded amino acid comprising an alkyne moiety and the water soluble polymer to be attached to the amino acid comprises wherein n is 1 - 10 ; X can be O , N , S or not present, Ph is an azide moiety . Alternatively , the converse reaction ( i. e ., phenyl, and W is a water soluble polymer . with the azide moiety on the amino acid and the alkyne moiety present on the water soluble polymer ) can also be Exemplary alkyne -containing amino acids can be repre performed . sented as follows: The azide functional group can also be reacted selectively » with a water soluble polymer containing an aryl ester and (CH2 ) R1X (CH2 ) mCCH appropriately functionalized with an aryl phosphine moiety to generate an amide linkage . The aryl phosphine group reduces the azide in situ and the resulting amine then reacts R2HN COR3 efficiently with a proximal ester linkage to generate the 40 corresponding amide . See , e . g . , E . Saxon and C . Bertozzi , wherein n is 0 - 10 : R , is an alkyl, aryl, substituted alkyl, or Science 287 , 2007 - 2010 (2000 ) . The azide -containing amino substituted aryl or not present ; X is O , N , S or not present; acid can be either an alkyl azide ( including but not limited m is 0 - 10 , R2 is H , an amino acid , a polypeptide, or an amino to , 2 - amino - 6 -azido - 1 -hexanoic acid ) or an aryl azide 45 terminus modification group , and Rz is H , an amino acid , a ( p - azido - phenylalanine ). polypeptide, or a carboxy terminus modification group . In Exemplary water soluble polymers containing an aryl some embodiments , n is 1 , R , is phenyl, X is not present, m ester and a phosphine moiety can be represented as follows: is 0 and the acetylene moiety is positioned in the para position relative to the alkyl side chain . In some embodi 50 ments, n is 1 , R is phenyl, X is O , m is 1 and the propargyloxy group is positioned in the para position rela tive to the alkyl side chain (i . e . , O - propargyl- tyrosine ). In some embodiments , n is 1 , R , and X are not present and m PPh2 is 0 ( i. e ., proparylglycine ) . 55 Alkyne- containing amino acids are commercially avail wherein X can be O , N , S or not present, Ph is phenyl, W is able . For example , propargylglycine is commercially avail a water soluble polymer and R can be H , alkyl, aryl, able from Peptech (Burlington , Mass . ) . Alternatively , substituted alkyl and substituted aryl groups . Exemplary R alkyne -containing amino acids can be prepared according to groups include but are not limited to — CH , , - C (CH ) , standard methods . For instance , p -propargyloxyphenylala - OR ', - NR ' R " , — SR ', -halogen , - C ( O ) R ', CONR ' R " , 60 nine can be synthesized , for example , as described in - S ( O ) , R ', - S ( O ) NR 'R " , CN and — NO2. R ', R " , R '" Deiters , A ., et al ., J . Am . Chem . Soc . 125 : 11782- 11783 and R " " each independently refer to hydrogen , substituted or (2003 ) , and 4 - alkynyl- L -phenylalanine can be synthesized unsubstituted heteroalkyl, substituted or unsubstituted aryl, as described in Kayser, B . , et al. , Tetrahedron 53 ( 7 ) : 2475 including but not limited to , aryl substituted with 1 - 3 halo - 2484 ( 1997 ) . Other alkyne -containing amino acids can be gens , substituted or unsubstituted alkyl, alkoxy or thio - 65 prepared by one of ordinary skill in the art . alkoxy groups, or arylalkyl groups. When a compound of the Exemplary azide - containing amino acids can be repre invention includes more than one R group , for example , sented as follows: US 9 , 962 ,450 B2 88 amino acids , including but not limited to , for incorporation (CH2 ) R1X (CH2 ) mNz into a protein . For example , the high charge density of a -amino acids suggests that these compounds are unlikely to be cell permeable . Natural amino acids are taken up into the R _ HN COR3 eukaryotic cell via a collection of protein - based transport systems. A rapid screen can be done which assesses which wherein n is 0 - 10 ; R , is an alkyl , aryl, substituted alkyl , unnatural amino acids , if any , are taken up by cells . See , e . g . , substituted aryl or not present ; X is O , N , S or not present; the toxicity assays in , e . g ., U . S . Patent Publication No . US m is 0 - 10 ; R , is H , an amino acid , a polypeptide, or an amino 2004 /0198637 entitled “ Protein Arrays " which is incorpo terminus modification group , and Rz is H , an amino acid , a 10 rated by reference herein ; and Liu , D . R . & Schultz , P . G . polypeptide, or a carboxy terminus modification group . In ( 1999 ) Progress toward the evolution of an organism with an some embodiments , n is 1 , R , is phenyl, X is not present, m expanded genetic code . PNAS United States 96 :4780 - 4785 . is 0 and the azide moiety is positioned para to the alkyl side . Although uptake is easily analyzed with various assays , an chain . In some embodiments , n is 0 - 4 and R , and X are not alternative to designing unnatural amino acids that are present, and m = 0 . In some embodiments , n is 1 , R , is 15 amenable to cellular uptake pathways is to provide biosyn phenyl, X is O , m is 2 and the B - azidoethoxy moiety is thetic pathways to create amino acids in vivo . positioned in the para position relative to the alkyl side Biosynthesis of Unnatural Amino Acids chain . Many biosynthetic pathways already exist in cells for the Azide -containing amino acids are available from com - production of amino acids and other compounds . While a mercial sources. For instance , 4 -azidophenylalanine can be 20 biosynthetic method for a particular unnatural amino acid obtained from Chem - Impex International, Inc . ( Wood Dale , may not exist in nature , including but not limited to , in a cell, Ill . ) . For those azide - containing amino acids that are not the invention provides such methods. For example , biosyn commercially available , the azide group can be prepared thetic pathways for unnatural amino acids are optionally relatively readily using standard methods known to those of generated in host cell by adding new enzymes or modifying ordinary skill in the art , including but not limited to , via 25 existing host cell pathways . Additional new enzymes are displacement of a suitable leaving group ( including but not optionally naturally occurring enzymes or artificially limited to , halide , mesylate , tosylate ) or via opening of a evolved enzymes . For example , the biosynthesis of p -amino suitably protected lactone . See, e . g ., Advanced Organic phenylalanine ( as presented in an example in WO 2002 / Chemistry by March ( Third Edition , 1985 , Wiley and Sons , 085923 entitled " In vivo incorporation of unnatural amino New York ). 30 acids " ) relies on the addition of a combination of known E . Aminothiol Reactive Groups enzymes from other organisms. The genes for these enzymes The unique reactivity of beta - substituted aminothiol func - can be introduced into a eukaryotic cell by transforming the tional groups makes them extremely useful for the selective cell with a plasmid comprising the genes . The genes , when modification of polypeptides and other biological molecules expressed in the cell , provide an enzymatic pathway to that contain aldehyde groups via formation of the thiazoli- 35 synthesize the desired compound . Examples of the types of dine . See , e . g . , J . Shao and J . Tam , J . Am . Chem . Soc . 1995 , enzymes that are optionally added are provided in the 117 ( 14 ) 3893 - 3899 . In some embodiments , beta -substituted examples below . Additional enzymes sequences are found , aminothiol amino acids can be incorporated into relaxin for example , in Genbank . Artificially evolved enzymes are polypeptides and then reacted with water soluble polymers also optionally added into a cell in the samemanner . In this comprising an aldehyde functionality . In some embodi- 40 manner, the cellular machinery and resources of a cell are ments , a water soluble polymer , drug conjugate or other manipulated to produce unnatural amino acids . payload can be coupled to a relaxin polypeptide comprising A variety of methods are available for producing novel a beta -substituted aminothiol amino acid via formation of enzymes for use in biosynthetic pathways or for evolution of the thiazolidine . existing pathways . For example , recursive recombination , F . Additional Reactive Groups 45 including but not limited to , as developed by Maxygen , Inc . Additional reactive groups and non - naturally encoded ( available on the World Wide Web at maxygen . com ) , is amino acids , including but not limited to para - amino -phe - optionally used to develop novel enzymes and pathways. nylalanine , that can be incorporated into relaxin polypep - See , e . g ., Stemmer ( 1994 ) , Rapid evolution of a protein in tides of the invention are described in the following patent vitro by DNA shuffling, Nature 370 ( 4 ) :389 - 391 ; and , Stem applications which are all incorporated by reference in their 50 mer , ( 1994 ) , DNA shuffling by random fragmentation and entirety herein : U . S . Patent Publication No . 2006 /0194256 , reassembly : In vitro recombination for molecular evolution , U . S . Patent Publication No. 2006 / 0217532 , U . S . Patent Proc . Natl . Acad . Sci. USA ., 91 : 10747 - 10751 . Similarly Publication No . 2006 /0217289 , U . S . Provisional Patent No. DesignPathTM , developed by Genencor ( available on the 60 /755 , 338 ; U . S . Provisional Patent No . 60 / 755 , 711 ; U . S . World Wide Web at genencor. com ) is optionally used for Provisional Patent No. 60 /755 ,018 ; International Patent 55 metabolic pathway engineering , including but not limited to , Application No . PCT/ US06 / 49397 ; WO 2006 / 069246 ; U . S . to engineer a pathway to create O - methyl- L - tyrosine in a Provisional Patent No . 60 /743 , 041; U . S . Provisional Patent cell. This technology reconstructs existing pathways in host No . 60 / 743 ,040 ; International Patent Application No. PCT ) organisms using a combination of new genes, including but US06 /47822 ; U . S . Provisional Patent No . 60 /882 ,819 ; U . S . not limited to , those identified through functional genomics , Provisional Patent No . 60 /882 ,500 ; and U . S . Provisional 60 and molecular evolution and design . Diversa Corporation Patent No . 60 /870 ,594 . These applications also discuss (available on the World Wide Web at diversa .com ) also reactive groups that may be present on PEG or other provides technology for rapidly screening libraries of genes polymers , including but not limited to , hydroxylamine ( ami and gene pathways , including but not limited to , to create nooxy ) groups for conjugation . new pathways. Cellular Uptake of Unnatural Amino Acids 65 Typically , the unnatural amino acid produced with an Unnatural amino acid uptake by a cell is one issue that is engineered biosynthetic pathway of the invention is pro typically considered when designing and selecting unnatural duced in a concentration sufficient for efficient protein US 9 , 962, 450 B2 89 90 biosynthesis , including but not limited to , a natural cellular to , there can be 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , or 10 ormore different amount, but not to such a degree as to affect the concentra - sites in the protein that comprise 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , or tion of the other amino acids or exhaust cellular resources. 10 or more different unnatural amino acids . In another Typical concentrations produced in vivo in this manner are aspect, a composition includes a protein with at least one , about 10 mM to about 0 .05 mM . Once a cell is transformed 5 but fewer than all , of a particular amino acid present in the with a plasmid comprising the genes used to produce protein is substituted with the unnatural amino acid . For a enzymes desired for a specific pathway and an unnatural amino acid is generated , in vivo selections are optionally given protein with more than one unnatural amino acids, the used to further optimize the production of the unnatural unnatural amino acids can be identical or different (includ amino acid for both ribosomal protein synthesis and cell 10 ing but not limited to , the protein can include two or more growth . different types of unnatural amino acids, or can include two Polypeptides with Unnatural Amino Acids of the same unnatural amino acid ) . For a given protein with The incorporation of an unnatural amino acid can be done more than two unnatural amino acids, the unnatural amino for a variety of purposes , including but not limited to , acids can be the same, different or a combination of a tailoring changes in protein structure and /or function , chang - 15 multiple unnatural amino acid of the same kind with at least ing size , acidity , nucleophilicity , hydrogen bonding , hydro one different unnatural amino acid . phobicity , accessibility of protease target sites, targeting to a Proteins or polypeptides of interest with at least one moiety ( including but not limited to , for a protein array ) , unnatural amino acid are a feature of the invention . The adding a biologically active molecule , attaching a polymer, invention also includes polypeptides or proteins with at least attaching a radionuclide, modulating serum half- life , modu - 20 one unnatural amino acid produced using the compositions lating tissue penetration ( e . g . tumors ) , modulating active and methods of the invention . An excipient ( including but transport , modulating tissue, cell or organ specificity or not limited to , a pharmaceutically acceptable excipient) can distribution , modulating immunogenicity , modulating pro - also be present with the protein . tease resistance , etc . Proteins that include an unnatural By producing proteins or polypeptides of interest with at amino acid can have enhanced or even entirely new catalytic 25 least one unnatural amino acid in eukaryotic cells , proteins or biophysical properties. For example , the following prop - or polypeptides will typically include eukaryotic post -trans erties are optionally modified by inclusion of an unnatural lational modifications. In certain embodiments , a protein amino acid into a protein : toxicity , biodistribution , structural includes at least one unnatural amino acid and at least one properties , spectroscopic properties, chemical and /or photo - post- translational modification that is made in vivo by a chemical properties , catalytic ability , half - life ( including but 30 eukaryotic cell , where the post -translational modification is not limited to , serum half - life ) , ability to react with other not made by a prokaryotic cell. For example , the post molecules , including but not limited to , covalently or non - translation modification includes , including but not limited covalently , and the like . The compositions including pro - to , acetylation , acylation , lipid -modification , palmitoylation , teins that include at least one unnatural amino acid are useful palmitate addition , phosphorylation , glycolipid - linkage for, including but not limited to , novel therapeutics , diag - 35 modification , glycosylation , and the like . In one aspect , the nostics , catalytic enzymes , industrial enzymes , binding pro - post- translational modification includes attachment of an teins ( including but not limited to , antibodies ), and including oligosaccharide (including but not limited to , (GlcNAc butnot limited to , the study of protein structure and function . Man ) 2 -Man - GlcNAc -GlcNAc )) to an asparagine by a Glc See, e . g . , Dougherty, ( 2000 ) Unnatural Amino Acids as NAC -asparagine linkage. See Table 1 which lists some Probes of Protein Structure and Function , Current Opinion 40 examples of N - linked oligosaccharides of eukaryotic pro in Chemical Biology , 4 :645 -652 . teins (additional residues can also be present, which are not In one aspect of the invention , a composition includes at shown ) . In another aspect, the post- translational modifica least one protein with at least one , including but not limited tion includes attachment of an oligosaccharide ( including to , at least two , at least three, at least four, at least five, at but not limited to , Gal -GalNAc , Gal -GlcNAc , etc . ) to a least six , at least seven , at least eight, at least nine, or at least 45 serine or threonine by a GalNAc -serine or GalNAc - threo ten or more unnatural amino acids . The unnatural amino nine linkage , or a GlcNAc -serine or a GlcNAc - threonine acids can be the same or different, including but not limited linkage . TABLE 1 Examples of oligosaccharides through GLCNAC - linkage

Type Base Structure

HIGH -MANNOSE Manal - 6 Mana1- 6 Manal - 3 > Manß1 - 4GlcNAcß1 - 4GlcNAcß1- Asn Mana1- 3 HYBRID Manal - 6 > Manß1 - 4GlcNAcß1 - 4GICNA B1- Asn GleNAcß1- 2 — Manal -3

COMPLEX GlcNAcß1 - 2 Manal - 6 Manß1 -4GlcNAcß1 -4GlcNAcß1 - Asn GlcNAcß1 - 2 -Manal - 3 US 9 , 962 , 450 B2 91 TABLE 1 -continued Examples of oligosaccharides through GLCNAC - linkage

Type Base Structure XYLOSE Manal - 6 > Manß1 - 4GlcNAcB1 - 4GlcNAcB1 - Asn Xy131 -2 10 In yet another aspect , the post - translation modification side chains can then be modified by , including but not includes proteolytic processing of precursors ( including but limited to , a Huisgen [ 3 + 2 ] cycloaddition reaction (see , e. g ., not limited to , calcitonin precursor, calcitonin gene - related Padwa, A . in Comprehensive Organic Synthesis , Vol. 4 , peptide precursor, preproparathyroid hormone , preproinsu - 15 ( 1991 ) Ed . Trost , B . M ., Pergamon , Oxford , p . 1069- 1109 ; lin , proinsulin , prepro - opiomelanocortin , pro - opiomelano and , Huisgen , R . in 1 ,3 - Dipolar Cycloaddition Chemistry , cortin and the like ), assembly into a multisubunit protein or ( 1984 ) Ed . Padwa, A ., Wiley , New York , p . 1 - 176 ) with , macromolecular assembly , translation to another site in the including but not limited to , alkynyl or azide derivatives, cell ( including but not limited to , to organelles , such as the respectively . Because this method involves a cycloaddition endoplasmic reticulum , the Golgi apparatus , the nucleus , 20 rather than a nucleophilic substitution , proteins can be lysosomes, peroxisomes , mitochondria , chloroplasts , vacu modified with extremely high selectivity . This reaction can oles , etc . , or through the secretory pathway ) . In certain be carried out at room temperature in aqueous conditions embodiments , the protein comprises a secretion or localiza - with excellent regioselectivity (1 , 4 > 1 , 5 ) by the addition of tion sequence , an epitope tag , a FLAG tag , a polyhistidine catalytic amounts of Cu ( I ) salts to the reaction mixture . See , tag , a GST fusion , or the like. 25 e . g . , Tornoe , et al. , ( 2002 ) J . Org . Chem . 67 : 3057 - 3064 ; and , One advantage of an unnatural amino acid is that it Rostovtsev , et al ., ( 2002 ) Angew . Chem . Int. Ed . 41: 2596 presents additional chemicalmoieties that can be used to add 2599 . Another method that can be used is the ligand additional molecules . These modifications can be made in exchange on a bisarsenic compound with a tetracysteine vivo in a eukaryotic or non - eukaryotic cell, or in vitro . Thus , in certain embodiments , the post - translational modification 30 motif , see, e. g ., Griffin , et al ., (1998 ) Science 281 :269 - 272 . is through the unnatural amino acid . For example , the A molecule that can be added to a protein of the invention post- translational modification can be through a nucleop through a [ 3 + 2 ] cycloaddition includes virtually any mol hilic -electrophilic reaction . Most reactions currently used ecule with an azide or alkynyl derivative . Molecules include , for the selective modification of proteins involve covalent but are not limited to , dyes, fluorophores , crosslinking bond formation between nucleophilic and electrophilic reac - 35. agentsage , saccharide derivatives , polymers ( including but not tion partners, including but not limited to the reaction of limited to , derivatives of polyethylene glycol) , photocross a -haloketones with histidine or cysteine side chains . Selec linkers , cytotoxic compounds, affinity labels , derivatives of tivity in these cases is determined by the number and biotin , resins, beads, a second protein or polypeptide (or accessibility of the nucleophilic residues in the protein . In more ), polynucleotide (s ) (including but not limited to , DNA , proteins of the invention , other more selective reactions can 40 RNA , etc . ), metal chelators, cofactors , fatty acids , carbohy be used such as the reaction of an unnatural keto - amino acid drates , and the like. These molecules can be added to an with hydrazides or aminooxy compounds, in vitro and in unnatural amino acid with an alkynyl group , including but vivo . See, e .g ., Cornish , et al. , ( 1996 ) J. Am . Chem . Soc ., not limited to , p -propargyloxyphenylalanine , or azido group , 118 :8150 - 8151 ; Mahal, et al . , ( 1997 ) Science , 276 : 1125 including but not limited to , p - azido -phenylalanine , respec 1128 ; Wang , et al. , (2001 ) Science 292: 498 -500 ; Chin , et al . , 45 tively . (2002 ) J . Am . Chem . Soc. 124 : 9026 - 9027 ; Chin , et al. , In Vivo Generation of Relaxin Polypeptides Comprising ( 2002 ) Proc . Natl . Acad . Sci. , 99 : 11020 - 11024 ; Wang , et al. , Non -Naturally - Encoded Amino Acids ( 2003 ) Proc . Natl. Acad . Sci. , 100 :56 -61 ; Zhang , et al. , The relaxin polypeptides of the invention can be gener ( 2003 ) Biochemistry, 42 :6735 - 6746 ; and , Chin , et al. , ated in vivo using modified tRNA and tRNA synthetases to ( 2003 ) Science , 301: 964 - 7 , all of which are incorporated by 50 add to or substitute amino acids that are not encoded in reference herein . This allows the selective labeling of vir - naturally -occurring systems. tually any protein with a host of reagents including fluoro - Methods for generating tRNAs and tRNA synthetases phores, crosslinking agents , saccharide derivatives and cyto - which use amino acids that are not encoded in naturally toxic molecules . See also , U . S . Pat. No . 6 , 927 , 042 entitled occurring systems are described in , e . g ., U . S . Pat. Nos. " Glycoprotein synthesis , ” which is incorporated by refer - 55 7 , 045 , 337 and 7 ,083 , 970 which are incorporated by refer ence herein . Post- translational modifications , including but ence herein . These methods involve generating a transla not limited to , through an azido amino acid , can also made tional machinery that functions independently of the syn through the Staudinger ligation ( including but not limited to , thetases and tRNAs endogenous to the translation system with triarylphosphine reagents ) . See , e . g ., Kiick et al. , ( and are therefore sometimes referred to as “ orthogonal" ) . ( 2002 ) Incorporation of azides into recombinant proteins for 60 Typically, the translation system comprises an orthogonal chemoselective modification by the Staudinger ligation , tRNA ( O - TRNA ) and an orthogonal aminoacyl tRNA syn PNAS 99 : 19 - 24 . thetase ( O -RS ) . Typically , the O -RS preferentially amino This invention provides another highly efficient method acylates the O - TRNA with at least one non -naturally occur for the selective modification of proteins, which involves the ring amino acid in the translation system and the O - RNA genetic incorporation of unnatural amino acids , including 65 recognizes at least one selector codon that is not recognized but not limited to , containing an azide or alkynylmoiety into by other tRNAs in the system . The translation system thus proteins in response to a selector codon . These amino acid inserts the non -naturally - encoded amino acid into a protein US 9 , 962 , 450 B2 93 94 produced in the system , in response to an encoded selector sequence using mutagenesis methods known in the art codon , thereby “ substituting ” an amino acid into a position ( including but not limited to , site - specific mutagenesis , in the encoded polypeptide. cassette mutagenesis , restriction selection mutagenesis , Awide variety of orthogonal tRNAs and aminoacyl tRNA etc . ) . synthetases have been described in the art for inserting 5 Methods for generating components of the protein bio particular synthetic amino acids into polypeptides , and are synthetic machinery , such as O - RSs, O -TRNAs , and orthogo generally suitable for use in the present invention . For n al O - TRNA/ O -RS pairs that can be used to incorporate a example , keto - specific O - TRNA / aminoacyl- tRNA syn non -naturally encoded amino acid are described in Wang , L . , thetases are described in Wang , L . , et al. , Proc . Natl. Acad . et al. , Science 292 : 498 - 500 ( 2001) ; Chin , J . W . , et al. , J . Am . Sci. USA 100 :56 -61 ( 2003 ) and Zhang, Z . et al. , Biochem . 10 Chem . Soc . 124 : 9026 - 9027 ( 2002) ; Zhang, Z . et al ., Bio 42 (22 ) :6735 - 6746 ( 2003 ). Exemplary O -RS , or portions chemistry 42 : 6735 -6746 ( 2003 ) .Methods and compositions thereof, are encoded by polynucleotide sequences and for the in vivo incorporation of non - naturally encoded amino include amino acid sequences disclosed in U . S . Pat. Nos. acids are described in U . S . Pat. No . 7 , 045 ,337 , which is 7 , 045 , 337 and 7 , 083 , 970 , each incorporated herein by ref- incorporated by reference herein . Methods for selecting an erence . Corresponding O - TRNA molecules for use with the 15 orthogonal tRNA -TRNA synthetase pair for use in vivo O - RSs are also described in U . S . Pat. Nos . 7 ,045 ,337 and translation system of an organism are also described in U . S . 7 , 083 , 970 which are incorporated by reference herein . Addi - Pat. Nos . 7 ,045 , 337 and 7 ,083 , 970 which are incorporated tional examples of O - tRNA /aminoacyl - tRNA synthetase by reference herein . PCT Publication No . WO 04 /035743 pairs are described in WO 2005 /007870 , WO 2005 / 007624 ; entitled “ Site Specific Incorporation of Keto Amino Acids and WO 2005 / 019415 . 20 into Proteins , " which is incorporated by reference herein in An example of an azide - specific O - TRNA / aminoacyl - its entirety , describes orthogonal RS and tRNA pairs for the tRNA synthetase system is described in Chin , J . W . , et al. , J . incorporation of keto amino acids. PCT Publication No . WO Am . Chem . Soc . 124 : 9026 - 9027 (2002 ). Exemplary O -RS 04 / 094593 entitled “ Expanding the Eukaryotic Genetic sequences for p -azido - L - Phe include , but are not limited to , Code , ” which is incorporated by reference herein in its nucleotide sequences SEQ ID NOs: 14 - 16 and 29 - 32 and 25 entirety , describes orthogonal RS and tRNA pairs for the amino acid sequences SEQ ID NOs: 46 -48 and 61 - 64 as incorporation of non - naturally encoded amino acids in disclosed in U . S . Pat . No . 7 ,083 , 970 which is incorporated eukaryotic host cells . by reference herein . Exemplary O - TRNA sequences suitable Methods for producing at least one recombinant orthogo for use in the present invention include , but are not limited nal aminoacyl- tRNA synthetase ( O - RS ) comprise : ( a ) gen to , nucleotide sequences SEQ ID NOs: 1 - 3 as disclosed in 30 erating a library of (optionally mutant) RSs derived from at U . S . Pat. No . 7 , 083 , 970 , which is incorporated by reference least one aminoacyl- tRNA synthetase (RS ) from a first herein . Other examples of O - TRNA / aminoacyl - tRNA syn - organism , including but not limited to , a prokaryotic organ thetase pairs specific to particular non - naturally encoded ism , such as Methanococcus jannaschii , Methanobacterium amino acids are described in U . S . Pat. No. 7 ,045 ,337 which thermoautotrophicum , Halobacterium , Escherichia coli, A . is incorporated by reference herein . O -RS and O - RNA that 35 fulgidus, P . furiosus , P . horikoshii, A . pernix , T . thermophi incorporate both keto - and azide - containing amino acids in lus , or the like, or a eukaryotic organism ; ( b ) selecting S . cerevisiae are described in Chin , J. W . , et al. , Science ( and / or screening ) the library of RSs ( optionally mutant 301: 964 -967 (2003 ) . RSs ) for members that aminoacylate an orthogonal tRNA Several other orthogonal pairs have been reported . Glu ( O - RNA ) in the presence of a non -naturally encoded amino taminyl ( see, e . g ., Liu , D . R . , and Schultz , P . G . ( 1999 ) Proc . 40 acid and a natural amino acid , thereby providing a pool of Natl . Acad . Sci . U . S . A 96 : 4780 -4785 ) , aspartyl ( see, e . g ., active (optionally mutant ) RSs ; and /or , ( c ) selecting (option Pastrnak , M . , et al. , ( 2000 ) Helv . Chim . Acta 83 : 2277 - 2286 ), ally through negative selection ) the pool for active RSS and tyrosyl (see , e . g . , Ohno , S ., et al. , ( 1998 ) J . Biochem . ( including but not limited to , mutantRSs ) that preferentially ( Tokyo , Jpn . ) 124 : 1065 - 1068 ; and , Kowal , A . K ., et al ., aminoacylate the O -TRNA in the absence of the non - natu ( 2001) Proc. Natl . Acad . Sci. U .S . A 98 : 2268 -2273 ) systems 45 rally encoded amino acid , thereby providing the at least one derived from S . cerevisiae tRNA ' s and synthetases have recombinant O - RS ; wherein the at least one recombinant been described for the potential incorporation of unnatural O -RS preferentially aminoacylates the O - TRNA with the amino acids in E . coli . Systems derived from the E . coli non - naturally encoded amino acid . glutaminyl ( see , e . g ., Kowal , A . K ., et al. , (2001 ) Proc . Natl. In one embodiment, the RS is an inactive RS . The inactive Acad . Sci. U . S . A 98 : 2268 - 2273 ) and tyrosyl (see , e . g ., 50 RS can be generated by mutating an active RS . For example , Edwards , H . , and Schimmel, P . ( 1990 ) Mol. Cell. Biol. the inactive RS can be generated by mutating at least about 10 : 1633 - 1641 ) synthetases have been described for use in S . 1 , at least about 2 , at least about 3 , at least about 4 , at least cerevisiae . The E . coli tyrosyl system has been used for the about 5 , at least about 6 , or at least about 10 or more amino incorporation of 3 - iodo - L - tyrosine in vivo , in mammalian acids to different amino acids , including but not limited to , cells . See , Sakamoto , K ., et al ., ( 2002 ) Nucleic Acids Res . 55 alanine . 30 :4692 -4699 . Libraries of mutant RSs can be generated using various Use of O - RNA / aminoacyl- tRNA synthetases involves techniques known in the art , including but not limited to selection of a specific codon which encodes the non - natu - rational design based on protein three dimensional RS rally encoded amino acid . While any codon can be used , it structure , or mutagenesis of RS nucleotides in a random or is generally desirable to select a codon that is rarely or never 60 rational design technique . For example , the mutant RSs can used in the cell in which the O - TRNA / aminoacyl- tRNA be generated by site -specific mutations , random mutations , synthetase is expressed . For example , exemplary codons diversity generating recombination mutations , chimeric con include nonsense codon such as stop codons ( amber, ochre , structs , rational design and by other methods described and opal ) , four or more base codons and other natural herein or known in the art . three -base codons that are rarely or unused . 65 In one embodiment, selecting ( and /or screening ) the Specific selector codon ( s ) can be introduced into appro - library of RSs ( optionally mutant RSs ) for members that are priate positions in the relaxin polynucleotide coding active, including but not limited to , that aminoacylate an US 9 , 962, 450 B2 95 96 orthogonal tRNA ( O - TRNA ) in the presence of a non selector codon (including but not limited to , a toxic marker naturally encoded amino acid and a natural amino acid , gene , including but not limited to , a ribonuclease barnase includes : introducing a positive selection or screening gene , comprising at least one selector codon ) , and the pool marker, including but not limited to , an antibiotic resistance of active (optionally mutant) RSs into a plurality of cells of gene, or the like, and the library of (optionally mutant) RSS 5 a second organism ; and identifying cells that survive or into a plurality of cells, wherein the positive selection and / or show a specific screening response in a first medium not screening marker comprises at least one selector codon , supplemented with the non -naturally encoded amino acid , including but not limited to , an amber , ochre , or opal codon ; but fail to survive or show a specific screening response in growing the plurality of cells in the presence of a selection a second medium supplemented with the non -naturally agent; identifying cells that survive ( or show a specific 10 encoded amino acid , thereby providing surviving or response ) in the presence of the selection and/ or screening screened cells with the at least one recombinant O -RS , agent by suppressing the at least one selector codon in the wherein the at least one recombinant O -RS is specific for the positive selection or screening marker, thereby providing a non - naturally encoded amino acid . In one aspect, the at least subset of positively selected cells that contains the pool of one selector codon comprises about two or more selector active (optionally mutant) RSs . Optionally , the selection 15 codons. Such embodiments optionally can include wherein and / or screening agent concentration can be varied . the at least one selector codon comprises two or more In one aspect, the positive selection marker is a chloram selector codons , and wherein the first and second organism phenicol acetyltransferase (CAT ) gene and the selector are different ( including but not limited to , each organism is codon is an amber stop codon in the CAT gene . Optionally , optionally , including but not limited to , a prokaryote , a the positive selection marker is a B - lactamase gene and the 20 eukaryote , a mammal, an Escherichia coli , a fungi, a yeast, selector codon is an amber stop codon in the B - lactamase an archaebacteria , a eubacteria , a plant, an insect, a protist, gene. In another aspect the positive screening marker com etc .) . Also , some aspects include wherein the negative prises a fluorescent or luminescent screening marker or an selection marker comprises a ribonuclease barnase gene affinity based screening marker ( including but not limited to , (which comprises at least one selector codon ) . Other aspects a cell surface marker ) . 25 include wherein the screening marker optionally comprises In one embodiment, negatively selecting or screening the a fluorescent or luminescent screening marker or an affinity pool for active RSS ( optionally mutants ) that preferentially based screening marker. In the embodiments herein , the aminoacylate the O - TRNA in the absence of the non - natu screenings and / or selections optionally include variation of rally encoded amino acid includes : introducing a negative the screening and / or selection stringency . selection or screening marker with the pool of active (op - 30 In one embodiment, the methods for producing at least tionally mutant) RSs from the positive selection or screening one recombinant orthogonal aminoacyl- tRNA synthetase into a plurality of cells of a second organism , wherein the (O - RS ) can further comprise : (d ) isolating the at least one negative selection or screening marker comprises at least recombinant O -RS ; ( e ) generating a second set of O -RS one selector codon ( including but not limited to , an antibi ( optionally mutated ) derived from the at least one recombi otic resistance gene , including but not limited to , a chloram - 35 nant O -RS ; and , ( f ) repeating steps ( b ) and ( c ) until a phenicol acetyltransferase (CAT ) gene ); and , identifying mutated O -RS is obtained that comprises an ability to cells that survive or show a specific screening response in a preferentially aminoacylate the O - TRNA . Optionally , steps firstmedium supplemented with the non - naturally encoded (d )- (f ) are repeated , including but not limited to , at least amino acid and a screening or selection agent, but fail to about two times. In one aspect, the second set of mutated survive or to show the specific response in a second medium 40 O -RS derived from at least one recombinant O -RS can be not supplemented with the non - naturally encoded amino generated by mutagenesis , including but not limited to , acid and the selection or screening agent, thereby providing random mutagenesis , site - specific mutagenesis , recombina surviving cells or screened cells with the at least one tion or a combination thereof. recombinant O - RS . For example , a CAT identification pro - The stringency of the selection /screening steps , including tocol optionally acts as a positive selection and/ or a negative 45 but not limited to , the positive selection / screening step ( b ) , screening in determination of appropriate O -RS recombi - the negative selection / screening step ( c ) or both the positive nants. For instance , a pool of clones is optionally replicated and negative selection / screening steps ( b ) and (c ) , in the on growth plates containing CAT (which comprises at least above - described methods , optionally includes varying the one selector codon ) either with or without one or more selection /screening stringency . In another embodiment, the non - naturally encoded amino acid . Colonies growing exclu - 50 positive selection screening step ( b ) , the negative selection sively on the plates containing non -naturally encoded amino screening step ( c ) or both the positive and negative selec acids are thus regarded as containing recombinant O -RS . In tion / screening steps ( b ) and (c ) comprise using a reporter, one aspect , the concentration of the selection (and / or screen wherein the reporter is detected by fluorescence -activated ing) agent is varied . In some aspects the first and second cell sorting (FACS ) or wherein the reporter is detected by organisms are different. Thus, the first and /or second organ - 55 luminescence . Optionally , the reporter is displayed on a cell ism optionally comprises: a prokaryote , a eukaryote , a surface, on a phage display or the like and selected based mammal , an Escherichia coli , a fungi, a yeast , an archae - upon affinity or catalytic activity involving the non -naturally bacterium , a eubacterium , a plant , an insect, a protist, etc . In encoded amino acid or an analogue . In one embodiment, the other embodiments , the screening marker comprises a fluo - mutated synthetase is displayed on a cell surface , on a phage rescent or luminescent screening marker or an affinity based 60 display or the like. screening marker . Methods for producing a recombinant orthogonal tRNA In another embodiment, screening or selecting ( including ( O -TRNA ) include: ( a ) generating a library of mutant tRNAS but not limited to , negatively selecting ) the pool for active derived from at least one tRNA , including but not limited to , ( optionally mutant ) RSs includes: isolating the pool of active a suppressor tRNA , from a first organism ; ( b ) selecting mutant RSs from the positive selection step ( b ) ; introducing 65 ( including but not limited to , negatively selecting ) or screen a negative selection or screening marker, wherein the nega ing the library for (optionally mutant) tRNAs that are tive selection or screening marker comprises at least one aminoacylated by an aminoacyl- tRNA synthetase (RS ) from US 9 , 962 , 450 B2 97 98 a second organism in the absence of a RS from the first the at least one recombinant tRNA is aminoacylated by the organism , thereby providing a pool of tRNAs (optionally O -RS and inserts an amino acid into a translation product mutant ) ; and , ( c ) selecting or screening the pool of tRNAs encoded by the positive marker gene , in response to the at ( optionally mutant) for members that are aminoacylated by least one selector codons. In another embodiment, the con an introduced orthogonal RS ( O -RS ) , thereby providing at 5 centration of the selection and / or screening agent is varied . least one recombinant O -tRNA ; wherein the at least one Methods for generating specific O - RNA / O -RS pairs are recombinant O -TRNA recognizes a selector codon and is not provided . Methods include : ( a ) generating a library of efficiency recognized by the RS from the second organism mutant tRNAs derived from at least one tRNA from a first and is preferentially aminoacylated by the O - RS . In some organism ; (b ) negatively selecting or screening the library embodiments the at least one tRNA is a suppressor tRNA 10 for ( optionally mutant ) tRNAs that are aminoacylated by an and / or comprises a unique three base codon of natural and / or aminoacyl -tRNA synthetase (RS ) from a second organism in unnatural bases, or is a nonsense codon , a rare codon , an the absence of a RS from the first organism , thereby pro unnatural codon , a codon comprising at least 4 bases , an viding a pool of ( optionally mutant ) tRNAS; ( c ) selecting or amber codon , an ochre codon , or an opal stop codon . In one screening the pool of (optionally mutant) tRNAs for mem embodiment, the recombinant O - TRNA possesses an 15 bers that are aminoacylated by an introduced orthogonal RS improvement of orthogonality . It will be appreciated that in ( O -RS ) , thereby providing at least one recombinant some embodiments, O - RNA is optionally imported into a O -URNA . The at least one recombinant O - TRNA recognizes first organism from a second organism without the need for a selector codon and is not efficiency recognized by the RS modification . In various embodiments, the first and second from the second organism and is preferentially aminoacy organisms are either the same or different and are optionally 20 lated by the O - RS . The method also includes ( d ) generating chosen from , including but not limited to , prokaryotes a library of (optionally mutant) RSs derived from at least one ( including but not limited to , Methanococcus jannaschii, aminoacyl -tRNA synthetase (RS ) from a third organism ; ( e ) Methanobacterium thermoautotrophicum , Escherichia coli, selecting or screening the library ofmutant RSs formembers Halobacterium , etc . ), eukaryotes, mammals , fungi, yeasts , that preferentially aminoacylate the at least one recombinant archaebacteria , eubacteria , plants , insects , protists , etc . 25 O - RNA in the presence of a non - naturally encoded amino Additionally, the recombinant tRNA is optionally amino acid and a natural amino acid , thereby providing a pool of acylated by a non - naturally encoded amino acid , wherein the active (optionally mutant) RSs ; and , ( f ) negatively selecting non -naturally encoded amino acid is biosynthesized in vivo or screening the pool for active (optionally mutant ) RSs that either naturally or through genetic manipulation . The non preferentially aminoacylate the at least one recombinant naturally encoded amino acid is optionally added to a 30 O -TRNA in the absence of the non - naturally encoded amino growth medium for at least the first or second organism . acid , thereby providing the at least one specific O -TRNA / O In one aspect , selecting ( including but not limited to , RS pair , wherein the at least one specific O - TRNA / O -RS pair negatively selecting ) or screening the library for (optionally comprises at least one recombinant O -RS that is specific for mutant) tRNAs that are aminoacylated by an aminoacyl the non -naturally encoded amino acid and the at least one tRNA synthetase (step ( b ) ) includes : introducing a toxic 35 recombinant O - RNA . Specific O -TRNA / O -RS pairs pro marker gene , wherein the toxic marker gene comprises at duced by the methods are included . For example , the spe least one of the selector codons ( or a gene that leads to the cific O -TRNA / O -RS pair can include , including but not production of a toxic or static agent or a gene essential to the limited to , a mutRNATyr -mutTyrRS pair , such as a organism wherein such marker gene comprises at least one mutRNATyr- SS12TyrRS pair , a mutRNALeu - mutLeuRS selector codon ) and the library of (optionally mutant) tRNAs 40 pair , a mutRNAThr -mutThrRS pair , a mutRNAGlu -mut into a plurality of cells from the second organism ; and , GluRS pair , or the like . Additionally , such methods include selecting surviving cells , wherein the surviving cells contain wherein the first and third organism are the same ( including the pool of (optionally mutant ) tRNAs comprising at least but not limited to , Methanococcus jannaschii ) . one orthogonal tRNA or nonfunctional tRNA . For example , Methods for selecting an orthogonal tRNA - TRNA syn surviving cells can be selected by using a comparison ratio 45 thetase pair for use in an in vivo translation system of a cell density assay. second organism are also included in the present invention . In another aspect, the toxic marker gene can include two The methods include : introducing a marker gene , a tRNA or more selector codons . In another embodiment of the and an aminoacyl- tRNA synthetase (RS ) isolated or derived methods , the toxic marker gene is a ribonuclease barnase from a first organism into a first set of cells from the second gene , where the ribonuclease barnase gene comprises at least 50 organism ; introducing the marker gene and the tRNA into a one amber codon . Optionally , the ribonuclease barnase gene duplicate cell set from a second organism ; and , selecting for can include two or more amber codons . surviving cells in the first set that fail to survive in the In one embodiment , selecting or screening the pool of duplicate cell set or screening for cells showing a specific ( optionally mutant) tRNAs for members that are aminoacy - screening response that fail to give such response in the lated by an introduced orthogonal RS ( O - RS ) can include : 55 duplicate cell set , wherein the first set and the duplicate cell introducing a positive selection or screening marker gene , set are grown in the presence of a selection or screening wherein the positive marker gene comprises a drug resis - agent, wherein the surviving or screened cells comprise the tance gene ( including but not limited to , O - lactamase gene , orthogonal tRNA -TRNA synthetase pair for use in the in the comprising at least one of the selector codons, such as at in vivo translation system of the second organism . In one least one amber stop codon ) or a gene essential to the 60 embodiment, comparing and selecting or screening includes organism , or a gene that leads to detoxification of a toxic an in vivo complementation assay . The concentration of the agent, along with the O -RS , and the pool of (optionally selection or screening agent can be varied . mutant) tRNAs into a plurality of cells from the second The organisms of the present invention comprise a variety organism ; and , identifying surviving or screened cells grown of organism and a variety of combinations . For example , the in the presence of a selection or screening agent , including 65 first and the second organisms of the methods of the present but not limited to , an antibiotic , thereby providing a pool of invention can be the same or different. In one embodiment, cells possessing the at least one recombinant tRNA , where the organisms are optionally a prokaryotic organism , includ US 9 , 962 , 450 B2 99 100 ing but not limited to , Methanococcus jannaschii , Metha library selections (Dahiyat and Mayo , Protein Sci 5 ( 5 ) : nobacterium thermoautotrophicum , Halobacterium , dos895 - 903 ( 1996 ) ; Dahiyat and Mayo , Science 278 (5335 ): Escherichia coli, A . fulgidus, P. furiosus, P . horikoshii, A . 82 - 7 ( 1997 ); Desjarlais and Handel , Protein Science 4 : pernix , T . thermophilus , or the like . Alternatively , the organ 2006 -2018 (1995 ) ; Harbury et al , PNAS USA 92 ( 18 ): 8408 isms optionally comprise a eukaryotic organism , including 5 8412 ( 1995 ) ; Kono et al. , Proteins: Structure, Function and but not limited to , plants ( including but not limited to , Genetics 19 : 244 - 255 ( 1994 ) ; Hellinga and Richards , PNAS complex plants such as monocots , or dicots ), algae , protists , USA 91: 5803 - 5807 (1994 ) ); and residue pair potentials fungi ( including but not limited to , yeast, etc ) , animals (Jones , Protein Science 3 : 567 - 574 , (1994 )) , and rational ( including but not limited to , mammals , insects , arthropods , design using Protein Design Automation® technology . (See etc . ) , or the like . In another embodiment, the second organ - " U . S . Pat . Nos . 6 , 188 , 965; 6 ,269 , 312 ; 6 , 403 , 312 ; W098 / ism is a prokaryotic organism , including but not limited to , 47089 , which are incorporated by reference ) . Residues that Methanococcus jannaschii, Methanobacterium thermoauto are critical for relaxin bioactivity, residues that are involved trophicum , Halobacterium , Escherichia coli , A . fulgidus , with pharmaceutical stability, antibody epitopes, or receptor Halobacterium , P . furiosus , P . horikoshii, A . pernix , T. binding residues may be mutated . U . S . Pat . Nos . 5 , 580 ,723 ; thermophilus, or the like . Alternatively , the second organism 5 , 834 ,250 ; 6 ,013 ,478 ; 6 , 428 , 954 ; and 6 ,451 , 561 , which are can be a eukaryotic organism , including but not limited to , incorporated by reference herein , describe methods for the a yeast, a animal cell , a plant cell , a fungus , a mammalian systematic analysis of the structure and function of poly cell, or the like . In various embodiments the first and second peptides such as relaxin by identifying active domains which organisms are different. influence the activity of the polypeptide with a target sub Location of Non - Naturally - Occurring Amino Acids in 20 stance . Residues other than those identified as critical to Relaxin Polypeptides biological activity by alanine or homolog scanning muta The present invention contemplates incorporation of one genesis may be good candidates for substitution with a or more non - naturally - occurring amino acids into relaxin non -naturally encoded amino acid depending on the desired polypeptides. One or more non - naturally occurring amino a ctivity sought for the polypeptide . Alternatively , the sites acids may be incorporated at a particular position which > > identified as critical to biological activity may also be good does not disrupt activity of the polypeptide . This can be candidates for substitution with a non -naturally encoded achieved by making " conservative” substitutions, including amino acid , again depending on the desired activity sought but not limited to , substituting hydrophobic amino acids for the polypeptide . Another alternative would be to simply with hydrophobic amino acids , bulky amino acids for bulky make serial substitutions in each position on the polypeptide amino acids, hydrophilic amino acids for hydrophilic amino su chain with a non -naturally encoded amino acid and observe acids and / or inserting the non - naturally - occurring amino the effect on the activities of the polypeptide. It is readily acid in a location that is not required for activity . apparent to those of ordinary skill in the art that any means , A variety of biochemical and structural approaches can be technique , or method for selecting a position for substitution employed to select the desired sites for substitution with a with a non -natural amino acid into any polypeptide is non - naturally encoded amino acid within the Insulinpoly - » suitable for use in the present invention . peptide. It is readily apparent to those of ordinary skill in the The structure and activity of mutants of relaxin polypep art that any position of the polypeptide chain is suitable for tides that contain deletions can also be examined to deter selection to incorporate a non - naturally encoded amino acid , mine regions of the protein that are likely to be tolerant of and selection may be based on rational design or by random substitution with a non -naturally encoded amino acid . In a selection for any or no particular desired purpose . Selection to similar manner, protease digestion and monoclonal antibod of desired sites may be for producing a relaxin molecule ies can be used to identify regions of relaxin that are having any desired property or activity , including but not responsible for binding the relaxin receptor. Once residues limited to , agonists , super -agonists , inverse agonists , that are likely to be intolerant to substitution with non antagonists, receptor binding modulators , receptor activity naturally encoded amino acids have been eliminated , the modulators, dimer or multimer formation , no change to * > impact of proposed substitutions at each of the remaining activity or property compared to the native molecule , or positions can be examined . Models may be generated from manipulating any physical or chemical property of the the three -dimensional crystal structures of other relaxin polypeptide such as solubility , aggregation , or stability . For family members and relaxin receptors . Protein Data Bank example, locations in the polypeptide required for biological (PDB , available on the World Wide Web at rcsb .org ) is a activity of relaxin polypeptides can be identified using point centralized database containing three -dimensional structural mutation analysis , alanine scanning, saturation mutagenesis data of large molecules of proteins and nucleic acids . and screening for biological activity , or homolog scanning Models may be made investigating the secondary and ter methods known in the art . Other methods can be used to tiary structure of polypeptides, if three - dimensional struc identify residues for modification of relaxin polypeptides tural data is not available . Thus, those of ordinary skill in the include , but are not limited to , sequence profiling ( Bowie » art can readily identify amino acid positions that can be and Eisenberg , Science 253 ( 5016 ) : 164 - 70 , ( 1991) ) , rotamer substituted with non -naturally encoded amino acids.

Relaxin A Chain Residue # complex solvent accessibility chain A / B interface Residue SEQ ID Residue Mainchain Sidechain Residue Mainchain Sidechain Name NO : 1 Average Average Average Average Average Average LEU 2 .393927 2 .336841 2 . 451013 0 . 00913 0 .0182590 TYR WN 1 .05745 1 .412109 0 . 880121 0 .448218 0 . 164923 0 .589865 US 9, 962 ,450 B2 101 102 -continued Relaxin A Chain Residue# 1 complex solvent accessibility chain A / B interface Residue SEQ ID Residue Mainchain Sidechain Residue Mainchain Sidechain Name NO : 1 Average Average Average Average Average Average SER 2 .039557 1 .726208 2 .666253 0 .094701 0 . 127334 0 .029435 ALA 1 .47603 1 . 32887 2 .064669 0 .040922 0 .051152 0 LEU 0 .520247 0 .621696 0 .418798 0 . 174242 0 . 189235 0 . 159248 ALA 0 . 476043 0 .450361 0 . 578769 0 . 512186 0 . 474717 0 . 662064 ASN 1 . 252271 0 .865912 1 .638629 0 . 32958 0 . 460124 0 . 199036 LYS 0 . 886439 0 .727133 1 .013884 0 .424786 0 . 531522 0 . 339397 CYS 0 . 307912 0 . 414588 0 .094561 0 . 731348 0 . 802621 0 . 588801 CYS 0 . 791671 0 . 774527 0 . 825959 1 . 267628 1 . 170385 1 . 462113 HIS 1 .644891 1 . 389182 1 .815363 0 .902367 0 .910146 0 . 897182 VAL 1 .432029 1 . 329367 1 .568911 0 . 865782 1 . 041144 0 .631966 GLY 0 . 721258 0 . 7212580 0 . 8141790. 8141790 CYS 0 .405285 0 . 470822 0 . 274212 0 . 79239 0 . 856379 0 .664412 THR 0 .511268 0 . 367613 0 .702808 1 . 096358 1 . 011865 1 . 209014 LYS AWNFOTOOvauAWNAOOOOvaaA 0 . 376592 0 .233542 0 .491032 2 . 691742 1 .414171 3 .713799 ARG 0 . 97893 0 . 478638 1 . 264811 1 .626733 1 . 12903 1 . 911135 SER 0 . 416177 0 . 424198 0 . 405482 0 . 476934 0 . 49423 0 . 453873 LEU 0 . 155542 0 . 267306 0 . 043778 0 . 735924 0 . 711222 0 .760627 ALA 0 .478645 0 . 507787 0 . 362075 1 . 105314 1 .045545 1 . 344386 ARG 1 .273184 1 . 102102 1 . 370945 0 . 551495 0 . 572567 0 .539454 PHE 1 .054989 1 . 153619 0 .998629 0 . 428106 0 .829454 0 . 198764 CYS 1 . 352353 1. 506838 1 .146374 2 .074392 .22276 1 .876563

Relaxin B Chain Residue # 1 complex solvent accessibility chain A / B interface Residue SEQ ID Residue Mainchain Sidechain Residue Mainchain Sidechain Name NO : 2 Average Average Average Average Average Average SER 2 .241497 2 . 362674 1 . 999142 1 . 249697 1 . 108926 1 . 531239 TRP 1 .036157 1 . 320435 0 . 922445 0 .774516 1 .009561 0 .680498 MET 0 .765903 0 . 783709 0 . 748097 1 . 378683 1 . 318601 1 . 438766 GLU 1 .401968 1 . 102195 1 .641786 1 .60857 1 . 38927 1. 784009 GLU 1 . 209446 0 . 935167 1 .428869 0 . 994341 1 . 251666 0 . 788481 VAL 1 . 123345 1 .08403 1 . 175766 1 . 917498 1 .613757 2 . 322484 ILE 0 . 478044 0 .594216 0 . 361873 1 . 329975 1 . 726264 0 . 933686 LYS 1 . 135226 0 . 723724 1 .464427 2 . 332893 1 .427852 3 .056925 LEU 0 . 579 0 . 504927 0 .653072 0 . 906043 1 . 195675 0 . 616411 CYS = 0 . 862862 0 .806072 0 . 976442 1 .030881 0 .795964 1 .500714 GLY = 1 .089858 1 .0898580 0 .604306 0 .604306 0 ARG = 3 .079311 1 .482092 3 . 992007 0 .024047 0 . 066128 0 GLU = 1 .46883 0 . 992251 1. 680643 0 . 137036 0 . 204496 0 . 107054 LEU = 0 . 378917 0 .508368 0 . 249467 0 .743117 0 .470105 1 .016129 VAL 1 .018006 0 . 820163 1 . 281796 0 . 279417 0 . 29034 0 . 264852 ARG va 1 .660023 0 . 937977 2 .072621 0 .048147 0 . 132403 0 ALA 0 .51055 0 .547669 0 . 362075 0 . 379088 0 . 387335 0 . 346101 GLN = 0 .436502 0 . 481692 0 . 400351 1 . 12185 0 . 733873 1 . 432231 ILE 1 .321723 0 . 993689 1 .649758 0 . 294635 0 .473331 0 . 115939 ALA 1 .001017 0 . 958569 1 . 170807 0 . 362917 0 .40431 0 . 197346 ILE 0 . 381207 0 .565133 0 . 197281 0 .689578 0 .665212 0 .713943 CYS 0 . 888302 0 . 931778 0 . 80135 0 . 782559 0 .685197 0 . 977284 GLY 1 .608804 1 . 608804 0 0 . 25224 0 .25224 0 MET 1 . 419412 1 . 489301 1 . 349524 0 . 239722 0 . 18572 0 .293724 SER 1 .07028 1 . 249672 0 .711497 0 . 582382 0 .433677 0 .879791 THR 2 . 199516 2 . 265998 2 . 110873 0 . 228179 0 . 155968 0 . 32446 TRP VOUAWN 4 . 74167 3 .651826 4 .971111 0

In some embodiments , the relaxin polypeptides of the of the exposed faces, may be a site or sites that are invention comprise one or more non - naturally encoded juxtaposed to a second relaxin , or other molecule or frag amino acids positioned in a region of the protein that does 60 ment thereof, may be in regions that are highly flexible , or not disrupt the structure of the polypeptide . structurally rigid , as predicted by the three - dimensional, Exemplary residues of incorporation of a non -naturally secondary , tertiary , or quaternary structure of relaxin , bound encoded amino acid may be those that are excluded from or unbound to its receptor, or coupled or not coupled to potential receptor binding regions, may be fully or partially another biologically active molecule , or may modulate the solvent exposed , have minimal or no hydrogen -bonding 65 conformation of the relaxin itself or a dimer or multimer interactions with nearby residues, may be minimally comprising one or more relaxin , by altering the flexibility or exposed to nearby reactive residues, may be on one or more rigidity of the complete structure as desired . US 9 , 962 ,450 B2 103 104 One of ordinary skill in the art recognizes that such ety , an actinic radiation excitable moiety , a photoisomeriz analysis of relaxin enables the determination ofwhich amino able moiety , biotin , a derivative of biotin , a biotin analogue, acid residues are surface exposed compared to amino acid a moiety incorporating a heavy atom , a chemically cleavable residues that are buried within the tertiary structure of the group , a photocleavable group , an elongated side chain , a protein . Therefore , it is an embodiment of the present 5 carbon - linked sugar, a redox - active agent, an amino thio invention to substitute a non -naturally encoded amino acid acid , a toxic moiety , an isotopically labeled moiety , a for an amino acid that is a surface exposed residue. biophysical probe, a phosphorescent group , a chemilumi In some embodiments , one or more non -naturally nescent group , an electron dense group , a magnetic group , encoded amino acids are incorporated in one or more of the an intercalating group , a chromophore, an energy transfer following positions in relaxin : in the A chain before position 10 agent, a biologically active agent, a detectable label, a small 1 ( i. e . at the N -terminus ) , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9, 10 , 11, 12 , molecule, a quantum dot, a nanotransmitter, a radionucle 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21, 22 (i . e ., at the carboxyl otide, a radiotransmitter, a neutron -capture agent, or any terminus of the protein ) , and any combination thereof (SEO combination of the above , or any other desirable compound ID NO : 1 ) or in the B chain before position 1 ( i . e . at the or substance ) that comprises a second reactive group . The N -terminus ), 1, 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9, 10 , 11, 12 , 13 , 14 , 15, 15 first reactive group reacts with the second reactive group to 16 , 17 , 18 , 19 , 20 , 21, 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 attach the molecule to the unnatural amino acid through a ( SEO ID NO : 2 ) . [ 3 + 2 ] cycloaddition . In one embodiment, the first reactive An examination of the crystal structure of relaxin and its group is an alkynyl or azido moiety and the second reactive interaction with the relaxin receptor can indicate which group is an azido or alkynyl moiety . For example , the first certain amino acid residues have side chains that are fully or 20 reactive group is the alkynyl moiety ( including but not partially accessible to solvent. The side chain of a non - limited to , in unnatural amino acid p - propargyloxyphenyl naturally encoded amino acid at these positions may point alanine ) and the second reactive group is the azido moiety . away from the protein surface and out into the solvent. In another example , the first reactive group is the azido In some embodiments , the non - naturally encoded amino moiety ( including but not limited to , in the unnatural amino acid at one or more of these positions is linked to a water 25 acid p - azido - L - phenylalanine ) and the second reactive soluble polymer , including but not limited to , positions: in group is the alkynyl moiety . the A chain before position 1 ( i . e . at the N -terminus ) , 1 , 2 , In some cases , the non - naturally encoded amino acid 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11, 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19, 20 , substitution ( s ) will be combined with other additions, sub 21 , 22 ( i . e . , at the carboxyl terminus of the protein ) , and any stitutions or deletions within the relaxin polypeptide to affect combination thereof (SEQ ID NO : 1 ) or in the B chain 30 other biological traits of the relaxin polypeptide . In some before position 1 ( i. e . at the N - terminus ) , 1 , 2 , 3 , 4 , 5 , 6 , 7 , cases , the other additions , substitutions or deletions may 8 , 9 , 10 , 11, 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , increase the stability ( including but not limited to , resistance 25 , 26 , 27 , 28 , 29 , 30 , 31 (SEQ ID NO : 2 ) . to proteolytic degradation ) of the relaxin polypeptide or A wide variety of non -naturally encoded amino acids can increase affinity of the relaxin polypeptide for its receptor. In be substituted for , or incorporated into , a given position in 35 some cases , the other additions, substitutions or deletions a relaxin polypeptide. In general, a particular non - naturally may increase the pharmaceutical stability of the relaxin encoded amino acid is selected for incorporation based on an polypeptide . In some cases , the other additions , substitutions examination of the three dimensional crystal structure of a or deletions may enhance the anti - viral activity of the relaxin relaxin polypeptide or other relaxin family member or polypeptide . In some cases, the other additions , substitutions relaxin analog with its receptor, a preference for conserva - 40 or deletions may increase the solubility ( including but not tive substitutions ( i . e ., aryl- based non -naturally encoded limited to , when expressed in E . coli or other host cells ) of amino acids , such as p -acetylphenylalanine or O - propargyl- the relaxin polypeptide . In some embodiments additions , tyrosine substituting for Phe, Tyr or Trp ) , and the specific substitutions or deletions may increase the relaxin polypep conjugation chemistry that one desires to introduce into the tide solubility following expression in E . coli or other relaxin polypeptide ( e . g . , the introduction of 4 - azidophenyl- 45 recombinant host cells . In some embodiments sites are alanine if one wants to effect a Huisgen 3 + 2 ] cycloaddition selected for substitution with a naturally encoded or non with a water soluble polymer bearing an alkyne moiety or a natural amino acid in addition to another site for incorpo amide bond formation with a water soluble polymer that ration of a non -natural amino acid that results in increasing bears an aryl ester that , in turn , incorporates a phosphine the polypeptide solubility following expression in E . coli or moiety ) 50 other recombinant host cells . In some embodiments , the In one embodiment, the method further includes incor - relaxin polypeptides comprise another addition , substitution porating into the protein the unnatural amino acid , where the or deletion thatmodulates affinity for the relaxin polypeptide unnatural amino acid comprises a first reactive group , and receptor, binding proteins , or associated ligand, modulates contacting the protein with a molecule ( including but not signal transduction after binding to the relaxin receptor, limited to , a label, a dye , a polymer, a water -soluble poly - 55 modulates circulating half - life , modulates release or bio mer, a derivative of polyethylene glycol, a photocrosslinker, availability , facilitates purification , or improves or alters a a radionuclide, a cytotoxic compound , a drug, an affinity particular route of administration . In some embodiments , the label , a photoaffinity label , a reactive compound , a resin , a relaxin polypeptides comprise an addition , substitution or second protein or polypeptide or polypeptide analog, an deletion that increases the affinity of the relaxin variant for antibody or antibody fragment, a metal chelator, a cofactor , 60 its receptor . Similarly , relaxin polypeptides can comprise a fatty acid , a carbohydrate , a polynucleotide , a DNA , a chemical or enzyme cleavage sequences , protease cleavage RNA , an antisense polynucleotide , a saccharide , a water - sequences , reactive groups, antibody -binding domains ( in soluble dendrimer , a cyclodextrin , an inhibitory ribonucleic cluding but not limited to , FLAG or poly - His ) or other acid , a biomaterial, a nanoparticle , a spin label, a fluoro - affinity based sequences ( including , but not limited to , phore , a metal- containing moiety , a radioactive moiety , a 65 FLAG , poly -His , GST, etc . ) or linked molecules ( including , novel functional group , a group that covalently or nonco - but not limited to , biotin ) that improve detection (including , valently interacts with other molecules, a photocaged moi- but not limited to , GFP ), purification , transport through US 9 , 962 , 450 B2 105 106 tissues or cell membranes, prodrug release or activation , more of the protein that comprises an unnatural amino acid , relaxin size reduction , or other traits of the polypeptide . or an amount that can be achieved with in vivo protein In some embodiments , the substitution of a non - naturally production methods ( details on recombinant protein produc encoded amino acid generates a relaxin antagonist . In some tion and purification are provided herein ) . In another aspect , embodiments , a non - naturally encoded amino acid is sub - 5 the protein is optionally present in the composition at a stituted or added in a region involved with receptor binding . concentration of, including but not limited to , at least 10 In some embodiments , relaxin antagonists comprise at least micrograms of protein per liter , at least 50 micrograms of one substitution that cause relaxin to act as an antagonist . In protein per liter, at least 75 micrograms of protein per liter, some embodiments , the relaxin antagonist comprises a non - at least 100 micrograms of protein per liter , at least 200 naturally encoded amino acid linked to a water soluble 10 micrograms of protein per liter , at least 250 micrograms of polymer that is present in a receptor binding region of the protein per liter, at least 500 micrograms of protein per liter, relaxin molecule . at least 1 milligram of protein per liter, or at least 10 In some cases , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , or more amino milligrams of protein per liter or more , in , including but not acids are substituted with one or more non -naturally - en - limited to , a cell lysate , a buffer, a pharmaceutical buffer , or coded amino acids . In some cases , the relaxin polypeptide 15 other liquid suspension ( including but not limited to , in a further includes 1, 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , or more volume of, including but not limited to , anywhere from substitutions of one or more non -naturally encoded amino about 1 nl to about 100 L or more ) . The production of large acids for naturally - occurring amino acids. For example , in quantities ( including but not limited to , greater that that some embodiments , one or more residues in relaxin are typically possible with other methods , including but not substituted with one or more non -naturally encoded amino 20 limited to , in vitro translation of a protein in a eukaryotic acids. In some cases , the one or more non - naturally encoded cell including at least one unnatural amino acid is a feature residues are linked to one or more lower molecular weight of the invention . linear or branched PEGs, thereby enhancing binding affinity eukaryotic host cell or non - eukaryotic host cell of the and comparable serum half - life relative to the species present invention provides the ability to biosynthesize pro attached to a single , higher molecular weight PEG . 25 teins that comprise unnatural amino acids in large useful In some embodiments , up to two of the following residues quantities. For example , proteins comprising an unnatural of relaxin are substituted with one or more non - naturally amino acid can be produced at a concentration of, including encoded amino acids . but not limited to , at least 10 ug/ liter, at least 50 ug / liter , at Expression in Non - Eukaryotes and Eukaryotes least 75 ug / liter, at least 100 ug / liter , at least 200 ug /liter , at To obtain high level expression of a cloned relaxin 30 least 250 ug / liter, or at least 500 ug / liter, at least 1 mg /liter , polynucleotide , one typically subclones polynucleotides at least 2 mg/ liter, at least 3 mg/ liter , at least 4 mg/ liter, at encoding a relaxin polypeptide of the invention into an least 5 mg/ liter , at least 6 mg/ liter , at least 7 mg/ liter , at least expression vector that contains a strong promoter to direct 8 mg/ liter, at least 9 mg/ liter, at least 10 mg / liter, at least 20 , transcription , a transcription / translation terminator, and if 30 , 40 , 50 , 60 , 70 , 80 , 90 , 100 , 200 , 300 , 400 , 500 , 600 , 700 , for a nucleic acid encoding a protein , a ribosome binding site 35 800 , 900 mg / liter, 1 g / liter, 5 g / liter , 10 g / liter or more of for translational initiation . Suitable bacterial promoters are protein in a cell extract, cell lysate , culture medium , a buffer, known to those of ordinary skill in the art and described , and /or the like . e . g . , in Sambrook et al. and Ausubel et al . A number of vectors suitable for expression of relaxin are Bacterial expression systems for expressing relaxin poly - commercially available . Useful expression vectors for peptides of the invention are available in , including but not 40 eukaryotic hosts , include but are not limited to , vectors limited to , E . coli, Bacillus sp . , Pseudomonas fluorescens, comprising expression control sequences from SV40 , Pseudomonas aeruginosa , Pseudomonas putida , and Salmo - bovine papilloma virus, adenovirus and cytomegalovirus . nella (Palva et al. , Gene 22 : 229 -235 ( 1983 ) ;Mosbach et al. , Such vectors include pCDNA3 . 1 ( + ) \Hyg ( Invitrogen , Carls Nature 302 : 543 - 545 ( 1983 ) ) . Kits for such expression sys - bad , Calif . , USA ) and PCI- neo ( Stratagene , La Jolla , Calif . , tems are commercially available . Eukaryotic expression 45 USA ) . Bacterial plasmids , such as plasmids from E . coli , systems for mammalian cells , yeast , and insect cells are including PBR322 , PET3a and pET12a , wider host range known to those of ordinary skill in the art and are also plasmids , such as RP4, phage DNAs, e . g . , the numerous commercially available. In cases where orthogonal tRNAs derivatives of phage lambda , e . g . , NM989 , and other DNA and aminoacyl tRNA synthetases (described above ) are used phages , such as M13 and filamentous single stranded DNA to express the relaxin polypeptides of the invention , host 50 phages may be used . The 2u plasmid and derivatives thereof, cells for expression are selected based on their ability to use the POT1 vector ( U . S . Pat. No . 4 ,931 , 373 which is incor the orthogonal components . Exemplary host cells include porated by reference ) , the PJSO37 vector described in (Ok Gram -positive bacteria ( including but not limited to B . kels , Ann . New York Aced . Sci. 782 , 202 207 , 1996 ) and brevis , B . subtilis , or Streptomyces ) and Gram -negative PPICZ A , B or C ( Invitrogen ) may be used with yeast host bacteria ( E . coli , Pseudomonas fluorescens, Pseudomonas 55 cells . For insect cells, the vectors include but are not limited aeruginosa , Pseudomonas putida ), as well as yeast and other to , pVL941 , pBG311 (Cate et al . , “ Isolation of the Bovine eukaryotic cells . Cells comprising O - RNA / O -RS pairs can and Human Genes for Mullerian Inhibiting Substance and be used as described herein . Expression of the Human Gene In Animal Cells ” , Cell, 45 , A eukaryotic host cell or non - eukaryotic host cell of the pp . 685 98 ( 1986 ) , pBluebac 4 . 5 and pMelbac ( Invitrogen , present invention provides the ability to synthesize proteins 60 Carlsbad , Calif . ) . that comprise unnatural amino acids in large useful quanti - The nucleotide sequence encoding a relaxin polypeptide ties . In one aspect , the composition optionally includes, may or may not also include sequence that encodes a signal including but not limited to , at least 10 micrograms, at least peptide . The signal peptide is present when the polypeptide 50 micrograms, at least 75 micrograms, at least 100 micro - is to be secreted from the cells in which it is expressed . Such grams, at least 200 micrograms, at least 250 micrograms, at 65 signal peptide may be any sequence . The signal peptide may least 500 micrograms, at least 1 milligram , at least 10 be prokaryotic or eukaryotic . Coloma, M ( 1992 ) J . Imm . milligrams, at least 100 milligrams, at least one gram , or Methods 152 :89 104 ) describe a signal peptide for use in US 9 , 962 , 450 B2 107 108 mammalian cells (murine Ig kappa light chain signal pep Of particular interest for use with the present invention tide ) . Other signal peptides include but are not limited to , the are species within the genera Pichia , Kluyveromyces , Sac a - factor signal peptide from S. cerevisiae (U .S . Pat. No. charomyces , Schizosaccharomyces , Hansenula , Torulopsis , 4 ,870 ,008 which is incorporated by reference herein ) , the and Candida , including , but not limited to , P . pastoris , P . signal peptide of mouse salivary amylase ( O . Hagenbuchle 5 guillerimondii , S . cerevisiae , S . carlsbergensis , S . diastati et al. , Nature 289 , 1981 , pp . 643 -646 ) , a modified carboxy cus, S . douglasii , S . kluyveri, S. norbensis , S . oviformis , K . peptidase signal peptide ( L . A . Valls et al. , Cell 48 , 1987 , pp . lactis , K . fragilis , C . albicans, C . maltosa , and H . polymor 887 - 897 ) , the yeast BAR1 signal peptide (WO 87 /02670 , pha . which is incorporated by reference herein ) , and the yeast The selection of suitable yeast for expression of relaxin aspartic protease 3 ( YAP3 ) signal peptide ( cf . M . Egel - 10 polypeptides is within the skill of one of ordinary skill in the Mitani et al. , Yeast 6 , 1990 , pp . 127 - 137 ) . art . In selecting yeast hosts for expression , suitable hosts Examples of suitable mammalian host cells are known to may include those shown to have , for example , good secre those of ordinary skill in the art. Such host cells may be tion capacity , low proteolytic activity , good secretion capac Chinese hamster ovary (CHO ) cells , ( e . g . CHO -K1 ; ATCC ity , good soluble protein production , and overall robustness . CCL -61 ) , Green Monkey cells (COS ) ( e . g . COS 1 ( ATCC 15 Yeast are generally available from a variety of sources CRL - 1650 ) , COS 7 ( ATCC CRL - 1651 ) ) ; mouse cells ( e . g . including , but not limited to , the Yeast Genetic Stock Center, NS / O ) , Baby Hamster Kidney (BHK ) cell lines ( e . g . ATCC Department of Biophysics and Medical Physics , University CRL - 1632 or ATCC CCL - 10 ), and human cells ( e . g . HEK of California (Berkeley , Calif. ) , and the American Type 293 ( ATCC CRL - 1573 ) ) , as well as plant cells in tissue Culture Collection (“ ATCC " ) (Manassas , Va . ) . culture . These cell lines and others are available from public 20 The term “ yeast host ” or “ yeast host cell” includes yeast depositories such as the American Type Culture Collection , that can be , or has been , used as a recipient for recombinant Rockville , Md. In order to provide improved glycosylation vectors or other transfer DNA . The term includes the prog of the relaxin polypeptide , a mammalian host cell may be eny of the original yeast host cell that has received the modified to express sialyltransferase , e . g . 1 , 6 - sialyltrans - recombinant vectors or other transfer DNA . It is understood ferase , e . g. as described in U . S . Pat . No . 5 ,047 , 335 , which is 25 that the progeny of a single parental cell may not necessarily incorporated by reference herein . be completely identical in morphology or in genomic or total Methods for the introduction of exogenous DNA into DNA complement to the original parent, due to accidental or mammalian host cells include but are not limited to , calcium deliberate mutation . Progeny of the parental cell that are phosphare -mediated transfection , electroporation , DEAE - sufficiently similar to the parent to be characterized by the dextran mediated transfection , liposome- mediated transfec - 30 relevant property , such as the presence of a nucleotide tion , viral vectors and the transfection methods described by sequence encoding a relaxin polypeptide , are included in the Life Technologies Ltd , Paisley, UK using Lipofectamin progeny intended by this definition . 2000 and Roche Diagnostics Corporation , Indianapolis , Expression and transformation vectors, including extra USA using FuGENE 6 . These methods are well known in the chromosomal replicons or integrating vectors , have been art and are described by Ausbel et al. ( eds . ) , 1996 , Current 35 developed for transformation into many yeast hosts . For Protocols in Molecular Biology , John Wiley & Sons, New example , expression vectors have been developed for S . York , USA . The cultivation of mammalian cells may be cerevisiae (Sikorski et al. , GENETICS ( 1989 ) 122: 19 ; Ito et performed according to established methods, e. g . as dis - al. , J. BACTERIOL . ( 1983 ) 153 : 163 ; Hinnen et al. , PROC . closed in (Animal Cell Biotechnology , Methods and Proto - NATL . ACAD . SCI. USA ( 1978 ) 75 : 1929 ) ; C . albicans cols , Edited by Nigel Jenkins, 1999 , Human Press Inc . 40 (Kurtz et al. , MOL . CELL . BIOL . ( 1986 ) 6 : 142 ) ; C . maltosa Totowa, N . J . , USA and Harrison Mass. and Rae I F , General (Kunze et al . , J . BASIC MICROBIOL . ( 1985 ) 25 : 141 ) ; H . Techniques of Cell Culture , Cambridge University Press polymorpha (Gleeson et al ., J . GEN . MICROBIOL . ( 1986 ) 1997 ) . 132: 3459 ; Roggenkamp et al ., MOL . GENETICS AND Expression Systems, Culture , and Isolation GENOMICS ( 1986 ) 202 : 302) ; K . fragilis (Das et al . , J . Relaxin polypeptides may be expressed in any number of 45 BACTERIOL . ( 1984 ) 158 : 1165 ) ; K . lactis (De Louvencourt suitable expression systems including , for example , yeast, et al. , J . BACTERIOL . ( 1983 ) 154 : 737 ; Van den Berg et al. , insect cells , mammalian cells , and bacteria . A description of BIOTECHNOLOGY (NY ) ( 1990 ) 8 : 135 ) ; P . guillerimondii exemplary expression systems is provided below . (Kunze et al. , J . BASIC MICROBIOL . ( 1985 ) 25 : 141 ) ; P . Yeast pastoris (U . S . Pat. Nos . 5 ,324 ,639 ; 4 , 929 ,555 ; and U . S . Pat. As used herein , the term “ yeast” includes any of the 50 No. 4 ,837 , 148 ; Cregg et al. , MOL . CELL . BIOL . ( 1985 ) various yeasts capable of expressing a gene encoding a 5 : 3376 ) ; Schizosaccharomyces pombe (Beach et al. , relaxin polypeptide . Such yeasts include , but are not limited NATURE ( 1982 ) 300 :706 ) ; and Y. lipolytica ; A . nidulans to , ascosporogenous yeasts ( Endomycetales ) , basidi - (Ballance et al ., BIOCHEM . BIOPHYS . RES . COMMUN . osporogenous yeasts and yeasts belonging to the Fungi (1983 ) 112: 284 -89 ; Tilburn et al. , GENE (1983 ) 26 :205 imperfecti ( Blastomycetes ) group . The ascosporogenous 55 221 ; and Yelton et al ., PROC . NATL . ACAD . SCI. USA yeasts are divided into two families , Spermophthoraceae and (1984 ) 81: 1470 - 74 ) ; A . niger (Kelly and Hynes, EMBO J. Saccharomycetaceae. The latter is comprised of four sub (1985 ) 4 :475 -479 ) ; T . reesia (EP O 244 234 ) ; and filamen families , Schizosaccharomycoideae ( e . g . , genus Schizosac tous fungi such as , e . g ., Neurospora , Penicillium , Tolypo charomyces) , Nadsonioideae, Lipomycoideae and Saccha - cladium (WO 91 /00357 ) , each incorporated by reference romycoideae ( e. g ., genera Pichia , Kluyveromyces and 60 herein . Saccharomyces ). The basidiosporogenous yeasts include the Control sequences for yeast vectors are known to those of genera Leucosporidium , Rhodosporidium , Sporidiobolus, ordinary skill in the art and include, but are not limited to , Filobasidium , and Filobasidiella . Yeasts belonging to the promoter regions from genes such as alcohol dehydrogenase Fungi Imperfecti (Blastomycetes ) group are divided into two ( ADH ) ( EP 0 284 044 ) ; enolase; glucokinase; glucose - 6 families, Sporobolomycetaceae ( e . g . , genera Sporobolomy - 65 phosphate isomerase ; glyceraldehyde - 3 -phosphate -dehydro ces and Bullera ) and Cryptococcaceae ( e . g ., genus Can - genase (GAP or GAPDH ) ; hexokinase ; phosphofructoki dida ). nase ; 3- phosphoglycerate mutase ; and pyruvate kinase US 9 , 962, 450 B2 109 110 (PyK ) (EP 0 329 203 ). The yeast PHO5 gene , encoding acid U . S . Reexamined Pat. Nos. RE37 ,343 and RE35 ,749 ; PCT phosphatase , also may provide useful promoter sequences Published Patent Applications WO 99 /07862 ; WO (Miyanohara et al. , PROC . NATL . ACAD . SCI. USA (1983 ) 98 /37208 ; and WO 98 /26080 ; European Patent Applications 80 : 1 ) . Other suitable promoter sequences for use with yeast EP O 946 736 ; EP 0 732 403 ; EP O 480 480 ; WO 90 / 10277 ; hosts may include the promoters for 3 -phosphoglycerate 5 EP 0 340 986 ; EP 0 329 203 ; EP 0 324 274 ; and EP 0 164 kinase (Hitzeman et al. , J. BIOL . CHEM . ( 1980 ) 255 : 556 . See also Gellissen et al. , ANTONIE VAN LEEUWEN 12073 ); and other glycolytic enzymes , such as pyruvate HOEK ( 1992 ) 62 ( 1 - 2 ) :79 - 93 ; Romanos et al. , YEAST decarboxylase , triosephosphate isomerase , and phosphoglu - ( 1992 ) 8 (6 ) : 423 - 488 ; Goeddel, METHODS IN ENZYMOL cose isomerase (Holland et al . , BIOCHEMISTRY ( 1978 ) OGY ( 1990 ) 185 : 3 - 7 , each incorporated by reference herein . 17 : 4900 ; Hess et al. , J . ADV. ENZYME REG . (1969 ) 10 The yeast host strains may be grown in fermentors during 7 : 149) . Inducible yeast promoters having the additional the amplification stage using standard feed batch fermenta advantage of transcription controlled by growth conditions tion methods known to those of ordinary skill in the art. The may include the promoter regions for alcohol dehydrogenase fermentation methods may be adapted to account for differ 2 ; isocytochrome C ; acid phosphatase ; metallothionein ; ences in a particular yeast host 's carbon utilization pathway glyceraldehyde - 3 - phosphate dehydrogenase ; degradative 15 or mode of expression control. For example , fermentation of enzymes associated with nitrogen metabolism ; and enzymes a Saccharomyces yeast host may require a single glucose responsible for maltose and galactose utilization . Suitable feed , complex nitrogen source ( e . g ., casein hydrolysates) , vectors and promoters for use in yeast expression are further and multiple vitamin supplementation . In contrast, the meth described in EP O 073 657 . ylotrophic yeast P . pastoris may require glycerol, methanol, Yeast enhancers also may be used with yeast promoters . 20 and trace mineral feeds, but only simple ammonium (nitro In addition , synthetic promoters may also function as yeast gen ) salts for optimal growth and expression . See, e . g . , U . S . promoters . For example , the upstream activating sequences Pat . No . 5 , 324 , 639 ; Elliott et al. , J . PROTEIN CHEM . (UAS ) of a yeast promoter may be joined with the tran - ( 1990 ) 9 : 95 ; and Fieschko et al. , BIOTECH . BIOENG . scription activation region of another yeast promoter, cre - ( 1987 ) 29 : 1113 , incorporated by reference herein . ating a synthetic hybrid promoter . Examples of such hybrid 25 Such fermentation methods, however , may have certain promoters include the ADH regulatory sequence linked to common features independent of the yeast host strain the GAP transcription activation region . See U .S . Pat . Nos . employed . For example, a growth limiting nutrient, typically 4 , 880 ,734 and 4 , 876 , 197 , which are incorporated by refer - carbon , may be added to the fermentor during the amplifi ence herein . Other examples of hybrid promoters include cation phase to allow maximal growth . In addition , fermen promoters that consist of the regulatory sequences of the 30 tation methods generally employ a fermentation medium ADH2, GAL4 , GAL10 , or PHO5 genes , combined with the designed to contain adequate amounts of carbon , nitrogen , transcriptional activation region of a glycolytic enzyme gene basal salts , phosphorus, and other minor nutrients ( vitamins, such as GAP or PyK . See EP 0 164 556 . Furthermore , a yeast trace minerals and salts , etc . ). Examples of fermentation promoter may include naturally occurring promoters of media suitable for use with Pichia are described in U . S . Pat. non -yeast origin that have the ability to bind yeast RNA 35 Nos . 5 , 324 ,639 and 5 , 231, 178 , which are incorporated by polymerase and initiate transcription . reference herein . Other control elements that may comprise part of the Baculovirus - Infected Insect Cells yeast expression vectors include terminators , for example , The term “ insect host" or " insect host cell ” refers to a from GAPDH or the enolase genes (Holland et al. , J . BIOL . insect that can be , or has been used as a recipient for CHEM . ( 1981 ) 256 : 1385 ) . In addition , the origin of repli - 40 recombinant vectors or other transfer DNA . The term cation from the 2u plasmid origin is suitable for yeast . A includes the progeny of the original insect host cell that has suitable selection gene for use in yeast is the trpl gene been transfected . It is understood that the progeny of a single present in the yeast plasmid . See Tschumper et al. , GENE parental cell may not necessarily be completely identical in ( 1980 ) 10 : 157 ; Kingsman et al. , GENE ( 1979 ) 7 : 141 . The morphology or in genomic or total DNA complement to the trpl gene provides a selection marker for a mutant strain of 45 original parent, due to accidental or deliberate mutation . yeast lacking the ability to grow in tryptophan . Similarly , Progeny of the parental cell that are sufficiently similar to the Leu2 - deficient yeast strains (ATCC 20, 622 or 38 ,626 ) are parent to be characterized by the relevant property , such as complemented by known plasmids bearing the Leu2 gene . the presence of a nucleotide sequence encoding a relaxin Methods of introducing exogenous DNA into yeast hosts polypeptide , are included in the progeny intended by this are known to those of ordinary skill in the art , and typically 50 definition . Baculovirus expression of relaxin polypeptides is include , but are not limited to , either the transformation of useful in the present invention and the use of rDNA tech spheroplasts or of intact yeast host cells treated with alkali n ology , polypeptides or precursors thereof because relaxin cations. For example , transformation of yeast can be carried may be biosynthesized in any number of host cells including out according to themethod described in Hsiao et al . , PROC . bacteria , mammalian cells , insect cells , yeast or fungi. An NATL . ACAD . SCI. USA ( 1979 ) 76 : 3829 and Van Solingen 55 embodiment of the present invention includes biosynthesis et al. , J .BACT . ( 1977 ) 130 : 946 . However, other methods for of relaxin , modified relaxin , relaxin polypeptides, or relaxin introducing DNA into cells such as by nuclear injection , analogs in bacteria , yeast or mammalian cells . Another electroporation , or protoplast fusion may also be used as embodiment of the present invention involves biosynthesis described generally in SAMBROOK ET AL . , MOLECU - done in E . coli or a yeast. Examples of biosynthesis in LAR CLONING : A LAB . MANUAL ( 2001 ) . Yeast host 60 mammalian cells and transgenic animals are described in cells may then be cultured using standard techniques known Hakola , K . [Molecular and Cellular Endocrinology , 127 : 59 to those of ordinary skill in the art. 69 , ( 1997 ) ] . Other methods for expressing heterologous proteins in The selection of suitable insect cells for expression of yeast host cells are known to those of ordinary skill in the relaxin polypeptides is known to those of ordinary skill in art. See generally U . S . Patent Publication No . 20020055169, 65 the art. Several insect species are well described in the art U . S . Pat . Nos. 6 , 361, 969 ; 6 ,312 , 923 ; 6 , 183 ,985 ; 6 ,083 ,723 ; and are commercially available including Aedes aegypti , 6 ,017 ,731 ; 5, 674 ,706 ; 5 ,629 , 203 ; 5 ,602 , 034 ; and 5 ,089 , 398 ; Bombyx mori , Drosophila melanogaster, Spodoptera fru US 9 , 962 , 450 B2 111 112 giperda , and Trichoplusia ni . In selecting insect hosts for moter to drive expression of heterologous genes . See gen expression , suitable hosts may include those shown to have , erally , O 'Reilly ET AL . , BACULOVIRUS EXPRESSION inter alia , good secretion capacity , low proteolytic activity , VECTORS : A LABORATORY MANUAL (1992 ) . and overall robustness. Insect are generally available from a Prior to inserting the foreign gene into the baculovirus variety of sources including , but not limited to , the Insect 5 genome, the above -described components, comprising a Genetic Stock Center, Department of Biophysics and Medi promoter, leader ( if desired ) , coding sequence of interest, cal Physics , University of California (Berkeley , Calif . ) ; and and transcription termination sequence , are typically the American Type Culture Collection (“ ATCC " ) (Manas assembled into an intermediate transplacement construct sas , Va . ) . ( transfer vector ). Intermediate transplacement constructs are Generally , the components of a baculovirus - infected 10 often maintained in a replicon , such as an extra chromo insect expression system include a transfer vector , usually a somal element ( e . g . , plasmids ) capable of stable mainte bacterial plasmid , which contains both a fragment of the nance in a host , such as bacteria . The replicon will have a baculovirus genome, and a convenient restriction site for replication system , thus allowing it to be maintained in a insertion of the heterologous gene to be expressed ; a wild suitable host for cloning and amplification . More specifi type baculovirus with sequences homologous to the bacu - 15 cally , the plasmid may contain the polyhedrin polyade lovirus -specific fragment in the transfer vector (this allows nylation signal (Miller , ANN . REV . MICROBIOL . ( 1988 ) for the homologous recombination of the heterologous gene 42: 177 ) and a prokaryotic ampicillin -resistance (amp ) gene in to the baculovirus genome ) ; and appropriate insect host and origin of replication for selection and propagation in E . cells and growth media . The materials , methods and tech - coli. niques used in constructing vectors , transfecting cells , pick - 20 One commonly used transfer vector for introducing for ing plaques , growing cells in culture , and the like are known eign genes into AcNPV is pAc373 . Many other vectors , in the art and manuals are available describing these tech - known to those of skill in the art, have also been designed niques . including, for example , pVL985 , which alters the polyhedrin After inserting the heterologous gene into the transfer start codon from ATG to ATT , and which introduces a vector , the vector and the wild type viral genome are 25 BamHI cloning site 32 base pairs downstream from the ATT. transfected into an insect host cell where the vector and viral See Luckow and Summers , VIROLOGY 170 :31 ( 1989 ) . genome recombine . The packaged recombinant virus is Other commercially available vectors include , for example , expressed and recombinant plaques are identified and puri - PBlueBac4 .5N /V5 -His ; pBlueBacHis2 ; pMelBac ; pBlue fied . Materials and methods for baculovirus/ insect cell Bac4 . 5 ( Invitrogen Corp . , Carlsbad , Calif. ) . expression systems are commercially available in kit form 30 After insertion of the heterologous gene , the transfer from , for example , Invitrogen Corp . (Carlsbad , Calif . ) . vector and wild type baculoviral genome are co - transfected These techniques are generally known to those of ordinary into an insect cell host . Methods for introducing heterolo skill in the art and fully described in SUMMERS AND gous DNA into the desired site in the baculovirus virus are SMITH , TEXAS AGRICULTURAL EXPERIMENT STA known in the art . See SUMMERS AND SMITH , TEXAS TION BULLETIN NO . 1555 ( 1987 ) , herein incorporated by 35 AGRICULTURAL EXPERIMENT STATION BULLETIN reference . See also , RICHARDSON , 39 METHODS IN NO . 1555 ( 1987) ; Smith et al ., MOL . CELL . BIOL . ( 1983 ) MOLECULAR BIOLOGY: BACULOVIRUS EXPRES - 3 : 2156 ; Luckow and Summers , VIROLOGY ( 1989 ) 170 :31 . SION PROTOCOLS ( 1995 ) ; AUSUBEL ET AL ., CUR - For example , the insertion can be into a gene such as the RENT PROTOCOLS IN MOLECULAR BIOLOGY 16 . 9 . polyhedrin gene, by homologous double crossover recom 16 . 11 ( 1994 ); KING AND POSSEE , THE BACULOVIRUS 40 bination ; insertion can also be into a restriction enzyme site SYSTEM : A LABORATORY GUIDE (1992 ) ; and engineered into the desired baculovirus gene. See Miller et O 'REILLY ET AL . , BACULOVIRUS EXPRESSION VEC - al. , BIOESSAYS ( 1989 ) 11 ( 4 ) :91 . TORS : A LABORATORY MANUAL ( 1992 ). Transfection may be accomplished by electroporation . Indeed , the production of various heterologous proteins See TROTTER AND WOOD , 39 METHODS IN using baculovirus/ insect cell expression systems is known to 45 MOLECULAR BIOLOGY ( 1995 ) ; Mann and King , J .GEN . those of ordinary skill in the art . See , e . g . , U . S . Pat. Nos . VIROL. ( 1989) 70 :3501 . Alternatively , liposomes may be 6 , 368 , 825 ; 6 , 342 , 216 ; 6 , 338 , 846 ; 6 , 261 , 805 ; 6 , 245 , 528 , used to transfect the insect cells with the recombinant 6 ,225 , 060 ; 6 , 183 , 987 ; 6 ,168 , 932 ; 6 , 126 ,944 ; 6 ,096 , 304 ; expression vector and the baculovirus . See , e . g ., Liebman et 6 , 013 , 433 ; 5 , 965 , 393 ; 5 , 939 ,285 ; 5 , 891, 676 ; 5 ,871 , 986 ; al. , BIOTECHNIQUES ( 1999 ) 26 ( 1 ) :36 ;Graves et al. , BIO 5 , 861, 279 ; 5 , 858 ,368 ; 5 , 843 ,733 ; 5 , 762 , 939 ; 5 , 753 ,220 ; 50 CHEMISTRY ( 1998 ) 37 :6050 ; Nomura et al. , J. BIOL . 5 ,605 ,827 ; 5 , 583 ,023 ; 5 ,571 , 709; 5 ,516 ,657 ; 5 , 290 ,686 ; CHEM . ( 1998 ) 273 (22 ) : 13570 ; Schmidt et al. , PROTEIN WO 02 / 06305 ; WO 01 /90390 ; WO 01/ 27301 ; WO EXPRESSION AND PURIFICATION ( 1998 ) 12 :323 ; 01/ 05956 ; WO 00 /55345 ; WO 00 / 20032 ; WO 99/ 51721 ; Siffert et al. , NATURE GENETICS ( 1998 ) 18 :45 ; TILKINS WO 99/ 45130 ; WO 99 /31257 ; WO 99 / 10515 ; WO ET AL . , CELL BIOLOGY: A LABORATORY HAND 99 /09193 ; WO 97 / 26332 ; WO 96 /29400 ; WO 96 / 25496 ; 55 BOOK 145 - 154 ( 1998 ) ; Cai et al. , PROTEIN EXPRES WO 96 / 06161 ; WO 95 /20672 ; WO 93 / 03173 ; WO SION AND PURIFICATION ( 1997 ) 10 : 263; Dolphin et al. , 92/ 16619; WO 92/ 02628 ; WO 92 /01801 ; WO 90 / 14428 ; NATURE GENETICS ( 1997) 17: 491; Kost et al ., GENE WO 90 / 10078 ; WO 90 /02566 ; WO 90 /02186 ; WO ( 1997 ) 190 : 139 ; Jakobsson et al. , J . BIOL . CHEM . ( 1996 ) 90 /01556 ; WO 89/ 01038 ; WO 89 /01037 ; WO 88 /07082 , 271: 22203 ; Rowles et al ., J. BIOL . CHEM . ( 1996 ) 271 (37 ) : which are incorporated by reference herein . 60 22376 ; Reverey et al . , J . BIOL . CHEM . ( 1996 ) 271 ( 39 ) : Vectors that are useful in baculovirus / insect cell expres 23607- 10 ; Stanley et al. , J. BIOL . CHEM . ( 1995 ) 270 :4121 ; sion systems are known in the art and include , for example , Sisk et al. , J. VIROL . ( 1994 ) 68 ( 2 ): 766 ; and Peng et al. , insect expression and transfer vectors derived from the BIOTECHNIQUES ( 1993 ) 14 ( 2 ): 274 . Commercially avail baculovirus Autographacalifornica nuclear polyhedrosis able liposomes include , for example , Cellfectin® and Lipo virus (ACNPV ) , which is a helper- independent , viral expres - 65 fectin® ( Invitrogen , Corp ., Carlsbad , Calif. ) . In addition , sion vector. Viral expression vectors derived from this calcium phosphate transfection may be used . See TROT system usually use the strong viral polyhedrin gene pro - TER AND WOOD , 39 METHODS IN MOLECULAR US 9 , 962 , 450 B2 113 114 BIOLOGY ( 1995 ) ; Kitts , NAR ( 1990 ) 18 ( 19 ) : 5667 ; and gene ) into mRNA . A promoter will have a transcription Mann and King , J. GEN . VIROL . ( 1989 ) 70 :3501 . initiation region which is usually placed proximal to the 5' Baculovirus expression vectors usually contain a baculo - end of the coding sequence . This transcription initiation virus promoter . A baculovirus promoter is any DNA region typically includes an RNA polymerase binding site sequence capable of binding a baculovirus RNA polymerase 5 and a transcription initiation site . A bacterial promoter may and initiating the downstream ( 3 ') transcription of a coding also have a second domain called an operator, that may sequence ( e . g ., structural gene ) into mRNA . A promoter will overlap an adjacent RNA polymerase binding site at which have a transcription initiation region which is usually placed RNA synthesis begins . The operator permits negative regu proximal to the 5 ' end of the coding sequence . This tran - lated ( inducible ) transcription , as a gene repressor protein scription initiation region typically includes an RNA poly - 10 may bind the operator and thereby inhibit transcription of a merase binding site and a transcription initiation site . A specific gene . Constitutive expression may occur in the baculovirus promoter may also have a second domain called absence of negative regulatory elements , such as the opera an enhancer, which , if present, is usually distal to the tor. In addition , positive regulation may be achieved by a structural gene. Moreover, expression may be either regu gene activator protein binding sequence, which , if present is lated or constitutive . 15 usually proximal (5 ') to the RNA polymerase binding Structural genes , abundantly transcribed at late times in sequence . An example of a gene activator protein is the the infection cycle , provide particularly useful promoter catabolite activator protein (CAP ) , which helps initiate tran sequences . Examples include sequences derived from the scription of the lac operon in Escherichia coli ( E . coli ) gene encoding the viral polyhedron protein (FRIESEN ET [Raibaud et al. , ANNU . REV. GENET. (1984 ) 18 : 173 ] . AL , The Regulation of Baculovirus Gene Expression in THE 20 Regulated expression may therefore be either positive or MOLECULAR BIOLOGY OF BACULOVIRUSES ( 1986 ) ; negative , thereby either enhancing or reducing transcription . EPO 127 839 and 0 155 476 ) and the gene encoding the p10 Sequences encoding metabolic pathway enzymes provide protein (Vlak et al. , J . GEN . VIROL . (1988 ) 69 :765 ) . particularly useful promoter sequences. Examples include The newly formed baculovirus expression vector is pack - promoter sequences derived from sugar metabolizing aged into an infectious recombinant baculovirus and subse - 25 enzymes , such as galactose , lactose (lac ) Chang et al. , quently grown plaques may be purified by techniques known NATURE ( 1977 ) 198 : 1056 ] , and maltose . Additional to those of ordinary skill in the art. See Miller et al. , examples include promoter sequences derived from biosyn BIOESSAYS (1989 ) 11 ( 4 ) :91 ; SUMMERS AND SMITH , thetic enzymes such as tryptophan ( trp ) [Goeddel et al . , TEXAS AGRICULTURAL EXPERIMENT STATION NUC. ACIDS RES . ( 1980 ) 8 :4057 ; Yelverton et al. , NUCL . BULLETIN NO . 1555 ( 1987 ) . 30 ACIDS RES . ( 1981 ) 9 : 731 ; U . S . Pat. No. 4 , 738 , 921 ; EP Recombinant baculovirus expression vectors have been Pub . Nos. 036 776 and 121 775 , which are incorporated by developed for infection into several insect cells . For reference herein ) . The B - galactosidase (bla ) promoter sys example , recombinant baculoviruses have been developed tem [Weissmann ( 1981 ) “ The cloning of interferon and other for, inter alia , Aedes aegypti ( ATCC No. CCL - 125 ) , Bombyx mistakes .” In Interferon 3 ( Ed . I . Gresser ) ] , bacteriophage mori (ATCC No. CRL -8910 ) , Drosophila melanogaster 35 lambda PL ( Shimatake et al ., NATURE ( 1981 ) 292 : 128 ) and ( ATCC No . 1963) , Spodoptera frugiperda , and Trichoplusia T5 [ U . S . Pat . No . 4 ,689 , 406 , which are incorporated by ni. See Wright, NATURE ( 1986 ) 321 :718 ; Carbonell et al ., reference herein ] promoter systems also provide useful J . VIROL . (1985 ) 56 : 153 ; Smith et al. , MOL . CELL . BIOL . promoter sequences . Preferred methods of the present inven ( 1983 ) 3 : 2156 . See generally , Fraser et al. , IN VITRO tion utilize strong promoters , such as the T7 promoter to CELL . DEV . BIOL . ( 1989 ) 25 : 225 . More specifically , the 40 induce relaxin polypeptides at high levels . Examples of such cell lines used for baculovirus expression vector systems vectors are known to those of ordinary skill in the art and commonly include , but are not limited to , Sf9 ( Spodoptera include the pET29 series from Novagen , and the pPOP frugiperda ) (ATCC No . CRL -1711 ) , Sf21 ( Spodoptera fru vectors described in W099 /05297 , which is incorporated by giperda ) ( Invitrogen Corp . , Cat. No. 11497 -013 ( Carlsbad , reference herein . Such expression systems produce high Calif . ) ) , Tri -368 ( Trichopulsia ni) , and High -FiveTM BTI - 45 levels of relaxin polypeptides in the host without compro TN - 5B1- 4 ( Trichopulsia ni) . mising host cell viability or growth parameters. pET19 Cells and culture media are commercially available for (Novagen ) is another vector known in the art. both direct and fusion expression of heterologous polypep . In addition , synthetic promoters which do not occur in tides in a baculovirus/ expression , and cell culture technol- nature also function as bacterial promoters . For example , ogy is generally known to those of ordinary skill in the art . 50 transcription activation sequences of one bacterial or bac E . Coli . Pseudomonas Species , and Other Prokaryotes teriophage promoter may be joined with the operon Bacterial expression techniques are known to those of sequences of another bacterial or bacteriophage promoter , ordinary skill in the art. A wide variety of vectors are creating a synthetic hybrid promoter [ U . S . Pat. No . 4 , 551, available for use in bacterial hosts. The vectors may be 433 , which is incorporated by reference herein ) . For single copy or low or high multicopy vectors . Vectors may 55 example , the tac promoter is a hybrid trp - lac promoter serve for cloning and / or expression . In view of the ample comprised of both trp promoter and lac operon sequences literature concerning vectors , commercial availability of that is regulated by the lac repressor [ Amann et al . , GENE manym vectors , and even manuals describing vectors and their ( 1983 ) 25 : 167 ; de Boer et al. , PROC . NATL . ACAD . SCI. restriction maps and characteristics, no extensive discussion ( 1983 ) 80 : 21 ] . Furthermore , a bacterial promoter can is required here . As is well -known , the vectors normally 60 include naturally occurring promoters of non -bacterial ori involve markers allowing for selection , which markers may gin that have the ability to bind bacterial RNA polymerase provide for cytotoxic agent resistance , prototrophy or immu - and initiate transcription . A naturally occurring promoter of nity . Frequently, a plurality of markers is present, which non -bacterial origin can also be coupled with a compatible provide for different characteristics . RNA polymerase to produce high levels of expression of A bacterial promoter is any DNA sequence capable of 65 some genes in prokaryotes . The bacteriophage T7 RNA binding bacterial RNA polymerase and initiating the down - polymerase /promoter system is an example of a coupled stream ( 3 ) transcription of a coding sequence ( e . g . structural promoter system [Studier et al. , J . MOL . BIOL . (1986 ) US 9 , 962 , 450 B2 115 116 189 : 113 ; Tabor et al ., Proc Natl. Acad . Sci. ( 1985 ) 82: 1074 ]. host cell and host cells with the proper expression construct In addition , a hybrid promoter can also be comprised of a are isolated ) , the recombinant host cell strain is cultured bacteriophage promoter and an E . coli operator region ( EP under conditions appropriate for production of relaxin poly Pub . No . 267 851) . peptides . As will be apparent to one of skill in the art, the In addition to a functioning promoter sequence, an effi - 5 method of culture of the recombinant host cell strain will be cient ribosome binding site is also useful for the expression dependent on the nature of the expression construct utilized of foreign genes in prokaryotes . In E . coli, the ribosome and the identity of the host cell . Recombinanthost strains are binding site is called the Shine - Dalgarno ( SD ) sequence and normally cultured using methods that are known to those of includes an initiation codon (ATG ) and a sequence 3 - 9 ordinary skill in the art. Recombinant host cells are typically nucleotides in length located 3 - 11 nucleotides upstream of 10 cultured in liquid medium containing assimilatable sources the initiation codon (Shine et al ., NATURE ( 1975) 254 : 34 ] . of carbon , nitrogen , and inorganic salts and , optionally , The SD sequence is thought to promote binding of mRNA containing vitamins, amino acids, growth factors , and other to the ribosome by the pairing of bases between the SD proteinaceous culture supplements known to those of ordi sequence and the 3 ' and of E . coli 16S rRNA [Steitz et al. nary skill in the art. Liquid media for culture of host cells " Genetic signals and nucleotide sequences in messenger 15 may optionally contain antibiotics or anti -fungals to prevent RNA ” , In Biological Regulation and Development: Gene the growth of undesirable microorganisms and / or com Expression ( Ed . R . F . Goldberger, 1979 ) ] . To express pounds including , but not limited to , antibiotics to select for eukaryotic genes and prokaryotic genes with weak ribo - host cells containing the expression vector. some -binding site [ Sambrook et al. “ Expression of cloned Recombinant host cells may be cultured in batch or genes in Escherichia coli ” , Molecular Cloning: A Labora - 20 continuous formats , with either cell harvesting in the case tory Manual, 1989 ). where the relaxin polypeptide accumulates intracellularly ) The term “ bacterial host ” or “ bacterial host cell” refers to or harvesting of culture supernatant in either batch or a bacterial that can be , or has been , used as a recipient for continuous formats. For production in prokaryotic host cells , recombinant vectors or other transfer DNA . The term batch culture and cell harvest are preferred . includes the progeny of the original bacterial host cell that 25 The relaxin polypeptides of the present invention are has been transfected . It is understood that the progeny of a normally purified after expression in recombinant systems. single parental cell may not necessarily be completely The relaxin polypeptide may be purified from host cells or identical in morphology or in genomic or total DNA comple - culture medium by a variety of methods known to the art . ment to the original parent, due to accidental or deliberate Relaxin polypeptides produced in bacterial host cells may be mutation . Progeny of the parental cell that are sufficiently 30 poorly soluble or insoluble ( in the form of inclusion bodies ) . similar to the parent to be characterized by the relevant In one embodiment of the present invention , amino acid property , such as the presence of a nucleotide sequence substitutions may readily be made in the relaxin polypeptide encoding a relaxin polypeptide, are included in the progeny that are selected for the purpose of increasing the solubility intended by this definition . of the recombinantly produced protein utilizing the methods The selection of suitable host bacteria for expression of 35 disclosed herein as well as those known in the art. In the case relaxin polypeptides is known to those of ordinary skill in of insoluble protein , the protein may be collected from host the art. In selecting bacterial hosts for expression , suitable cell lysates by centrifugation and may further be followed by hosts may include those shown to have , inter alia , good homogenization of the cells . In the case of poorly soluble inclusion body formation capacity , low proteolytic activity , protein , compounds including, but not limited to , polyeth and overall robustness . Bacterial hosts are generally avail - 40 ylene imine (PEI ) may be added to induce the precipitation able from a variety of sources including, but not limited to , of partially soluble protein . The precipitated protein may the BacterialGenetic Stock Center, Department of Biophys - then be conveniently collected by centrifugation . Recombi ics and Medical Physics, University of California (Berkeley , nant host cells may be disrupted or homogenized to release Calif .) ; and the American Type Culture Collection the inclusion bodies from within the cells using a variety of (" ATCC " ) (Manassas , Va . ) . Industrial/ pharmaceutical fer - 45 methods known to those of ordinary skill in the art . Host cell mentation generally use bacterial derived from K strains disruption or homogenization may be performed using well ( e . g . W3110 ) or from bacteria derived from B strains ( e. g . known techniques including, but not limited to , enzymatic BL21) . These strains are particularly useful because their cell disruption , sonication , dounce homogenization , or high growth parameters are extremely well known and robust. In pressure release disruption . In one embodiment of the addition , these strains are non -pathogenic , which is com - 50 method of the present invention , the high pressure release mercially important for safety and environmental reasons. technique is used to disrupt the E . coli host cells to release Other examples of suitable E . coli hosts include, but are not the inclusion bodies of the relaxin polypeptides . When limited to , strains of BL21 , DH10B , or derivatives thereof. handling inclusion bodies of relaxin polypeptide , it may be In another embodiment of the methods of the present advantageous to minimize the homogenization time on invention , the E . coli host is a protease minus strain includ - 55 repetitions in order to maximize the yield of inclusion bodies ing , but not limited to , OMP - and LON - . The host cell strain without loss due to factors such as solubilization , mechani may be a species of Pseudomonas, including but not limited cal shearing or proteolysis . to , Pseudomonas fluorescens, Pseudomonas aeruginosa , Insoluble or precipitated relaxin polypeptide may then be and Pseudomonas putida . Pseudomonas fluorescens biovar solubilized using any of a number of suitable solubilization 1, designated strain MB101 , is known to be useful for 60 agents known to the art. The relaxin polypeptide may be recombinant production and is available for therapeutic solubilized with urea or guanidine hydrochloride . The vol protein production processes . Examples of a Pseudomonas ume of the solubilized relaxin polypeptide should be mini expression system include the system available from The mized so that large batches may be produced using conve Dow Chemical Company as a host strain (Midland , Mich . niently manageable batch sizes . This factor may be available on the World Wide Web at dow .com ) . 65 significant in a large - scale commercial setting where the Once a recombinant host cell strain has been established recombinant host may be grown in batches that are thou ( i. e ., the expression construct has been introduced into the sands of liters in volume. In addition , when manufacturing US 9 , 962 , 450 B2 117 118 relaxin polypeptide in a large - scale commercial setting, in filtered to remove cellular debris. The supernatant may be particular for human pharmaceutical uses , the avoidance of concentrated or diluted to a desired volume or diafiltered harsh chemicals that can damage the machinery and con - into a suitable buffer to condition the preparation for further tainer, or the protein product itself , should be avoided , if purification . Further purification of the relaxin polypeptide possible . It has been shown in the method of the present 5 of the present invention includes separating deamidated and invention that the milder denaturing agent urea can be used clipped forms of the relaxin polypeptide variant from the to solubilize the relaxin polypeptide inclusion bodies in intact form . place of the harsher denaturing agent guanidine hydrochlo Any of the following exemplary procedures can be ride . The use of urea significantly reduces the risk of damage employed for purification of relaxin polypeptides of the to stainless steel equipment utilized in the manufacturing 10 invention : affinity chromatography ; anion - or cation - ex and purification process of relaxin polypeptide while effi - change chromatography (using , including but not limited to , ciently solubilizing the relaxin polypeptide inclusion bodies . DEAE SEPHAROSE ) ; chromatography on silica ; high per In the case of soluble relaxin protein , the relaxin may be formance liquid chromatography (HPLC ) ; reverse phase secreted into the periplasmic space or into the culture HPLC ; gel filtration ( using , including but not limited to , medium . In addition , soluble relaxin may be present in the 15 SEPHADEX G - 75 ) ; hydrophobic interaction chromatogra cytoplasm of the host cells . It may be desired to concentrate phy ; size - exclusion chromatography; metal- chelate chroma soluble relaxin prior to performing purification steps. Stan - tography ; ultrafiltration /diafiltration ; ethanol precipitation ; dard techniques known to those of ordinary skill in the art ammonium sulfate precipitation ; chromatofocusing ; dis may be used to concentrate soluble relaxin from , for placement chromatography ; electrophoretic procedures ( in example , cell lysates or culture medium . In addition , stan - 20 cluding but not limited to preparative isoelectric focusing ) , dard techniques known to those of ordinary skill in the art differential solubility ( including but not limited to ammo may be used to disrupt host cells and release soluble relaxin nium sulfate precipitation ) , SDS -PAGE , or extraction . from the cytoplasm or periplasmic space of the host cells. Proteins of the present invention , including but not lim When relaxin polypeptide is produced as a fusion protein , ited to , proteins comprising unnatural amino acids, peptides the fusion sequence may be removed . Removal of a fusion 25 comprising unnatural amino acids , antibodies to proteins sequence may be accomplished by enzymatic or chemical comprising unnatural amino acids, binding partners for cleavage . Enzymatic removal of fusion sequences may be proteins comprising unnatural amino acids , etc . , can be accomplished using methods known to those of ordinary purified , either partially or substantially to homogeneity , skill in the art . The choice of enzyme for removal of the according to standard procedures known to and used by fusion sequence will be determined by the identity of the 30 those of skill in the art . Accordingly, polypeptides of the fusion , and the reaction conditions will be specified by the invention can be recovered and purified by any of a number choice of enzyme as will be apparent to one of ordinary skill of methods known to those of ordinary skill in the art , in the art. Chemical cleavage may be accomplished using including but not limited to , ammonium sulfate or ethanol reagents known to those of ordinary skill in the art, including precipitation , acid or base extraction , column chromatogra but not limited to , cyanogen bromide , TEV protease , and 35 phy , affinity column chromatography , anion or cation other reagents . The cleaved relaxin polypeptide may be exchange chromatography, phosphocellulose chromatogra purified from the cleaved fusion sequence by methods phy , hydrophobic interaction chromatography, hydroxylapa known to those of ordinary skill in the art . Such methods will tite chromatography , lectin chromatography, gel electropho be determined by the identity and properties of the fusion resis and the like . Protein refolding steps can be used , as sequence and the relaxin polypeptide, as will be apparent to 40 desired , in making correctly folded mature proteins. High one of ordinary skill in the art. Methods for purification may performance liquid chromatography (HPLC ) , affinity chro include , but are not limited to , size- exclusion chromatogra matography or other suitable methods can be employed in phy, hydrophobic interaction chromatography, ion - exchange final purification steps where high purity is desired . In one chromatography or dialysis or any combination thereof. embodiment, antibodies made against unnatural amino acids The relaxin polypeptide may also be purified to remove 45 (or proteins or peptides comprising unnatural amino acids ) DNA from the protein solution . DNA may be removed by are used as purification reagents, including but not limited any suitable method known to the art, such as precipitation to , for affinity -based purification of proteins or peptides or ion exchange chromatography , but may be removed by comprising one or more unnatural amino acid ( s ) . Once precipitation with a nucleic acid precipitating agent , such as, purified , partially or to homogeneity , as desired , the poly but not limited to , protamine sulfate . The relaxin polypep - 50 peptides are optionally used for a wide variety of utilities , tide may be separated from the precipitated DNA using including but not limited to , as assay components , therapeu standard well known methods including, but not limited to , tics , prophylaxis , diagnostics , research reagents , and / or as centrifugation or filtration . Removal of host nucleic acid immunogens for antibody production . Antibodies generated molecules is an important factor in a setting where the against polypeptides of the present invention may be relaxin polypeptide is to be used to treat humans and the 55 obtained by administering the polypeptides or epitope -bear methods of the present invention reduce host cell DNA to ing fragments , or cells to an animal, preferably a non -human pharmaceutically acceptable levels . animal , using routine protocols . One of ordinary skill in the Methods for small -scale or large - scale fermentation can art could generate antibodies using a variety of known also be used in protein expression , including but not limited techniques. Also , transgenic mice , or other organisms, to , fermentors, shake flasks , fluidized bed bioreactors, hol- 60 including other mammals , may be used to express human low fiber bioreactors , roller bottle culture systems, and ized antibodies. The above - described antibodies may be stirred tank bioreactor systems. Each of these methods can employed to isolate or to identify clones expressing the be performed in a batch , fed -batch , or continuous mode polypeptide or to purify the polypeptides . Antibodies against process . polypeptides of the present invention may also be employed Human relaxin polypeptides of the invention can gener - 65 to treat diseases. ally be recovered using methods standard in the art. For Polypeptides and polynucleotides of the present invention example , culture medium or cell lysate can be centrifuged or may also be used as vaccines . Accordingly , in a further US 9 ,962 ,450 B2 119 120 aspect , the present invention relates to a method for inducing range of functional groups has been introduced into proteins an immunological response in a mammal that comprises for studies of protein stability , protein folding , enzyme inoculating the mammal with a polypeptide of the present mechanism , and signal transduction . invention , adequate to produce antibody and / or T cell In addition to other references noted herein , a variety of immune response , including , for example , cytokine -produc - 5 purification /protein folding methods are known to those of ing T cells or cytotoxic T cells , to protect said animal from ordinary skill in the art , including , but not limited to , those disease , whether that disease is already established within set forth in R . Scopes, Protein Purification , Springer- Verlag , the individual or not. An immunological response in a N . Y . (1982 ); Deutscher, Methods in Enzymology Vol. 182 : mammal may also be induced by a method comprises Guide to Protein Purification , Academic Press , Inc . N . Y . delivering a polypeptide of the present invention via a vector 10 ( 1990 ) ; Sandana , ( 1997) Bioseparation of Proteins , Aca directing expression of the polynucleotide and coding for the demic Press , Inc . ; Bollag et al. ( 1996 ) Protein Methods, 2nd polypeptide in vivo in order to induce such an immunologi Edition Wiley - Liss, NY ; Walker, ( 1996 ) The Protein Proto cal response to produce antibody to protect said animal from cols Handbook Humana Press , NJ, Harris and Angal, (1990 ) diseases of the invention . One way of administering the Protein Purification Applications : A Practical Approach IRL vector is by accelerating it into the desired cells as a coating 15 Press at Oxford , Oxford , England ; Harris and Angal , Protein on particles or otherwise. Such nucleic acid vector may Purification Methods: A Practical Approach IRL Press at comprise DNA , RNA , a modified nucleic acid , or a DNA Oxford , Oxford , England ; Scopes , ( 1993 ) Protein Purifica RNA hybrid . For use as a vaccine, a polypeptide or a nucleic tion : Principles and Practice 3rd Edition Springer Verlag , acid vector will be normally provided as a vaccine formu NY ; Janson and Ryden , ( 1998 ) Protein Purification : Prin lation ( composition ) . The formulation may further comprise 20 ciples , High Resolution Methods and Applications, Second a suitable carrier. Since a polypeptide may be broken down Edition Wiley -VCH , NY ; and Walker ( 1998 ), Protein Pro in the stomach , it may be administered parenterally ( for tocols on CD - ROM Humana Press , NJ; and the references instance , subcutaneous , intramuscular , intravenous, or intra - cited therein . dermal injection ) . Formulations suitable for parenteral One advantage of producing a protein or polypeptide of administration include aqueous and non -aqueous sterile 25 interest with an unnatural amino acid in a eukaryotic host injection solutions that may contain anti -oxidants , buffers , cell or non - eukaryotic host cell is that typically the proteins bacteriostats and solutes that render the formulation instonic or polypeptides will be folded in their native conformations . with the blood of the recipient; and aqueous and non - However, in certain embodiments of the invention , those of aqueous sterile suspensions that may include suspending skill in the art will recognize that , after synthesis, expression agents or thickening agents . The vaccine formulation may 30 and / or purification , proteins or peptides can possess a con also include adjuvant systems for enhancing the immuno - formation different from the desired conformations of the genicity of the formulation which are known to those of relevant polypeptides . In one aspect of the invention , the ordinary skill in the art. The dosage will depend on the expressed protein or polypeptide is optionally denatured and specific activity of the vaccine and can be readily deter then renatured . This is accomplished utilizing methods mined by routine experimentation . 35 known in the art , including but not limited to , by adding a Expression in Alternate Systems chaperonin to the protein or polypeptide of interest, by Several strategies have been employed to introduce solubilizing the proteins in a chaotropic agent such as unnatural amino acids into proteins in non -recombinant host guanidine HCl, utilizing protein disulfide isomerase , etc . cells , mutagenized host cells , or in cell- free systems. These In general, it is occasionally desirable to denature and systems are also suitable for use in making the Relaxin 40 reduce expressed polypeptides and then to cause the poly polypeptides of the present invention . Derivatization of peptides to re - fold into the preferred conformation . For amino acids with reactive side -chains such as Lys , Cys and example , guanidine , urea , DTT, DTE , and /or a chaperonin Tyr resulted in the conversion of lysine to N2 -acetyl - lysine . can be added to a translation product of interest . Methods of Chemical synthesis also provides a straightforward method reducing , denaturing and renaturing proteins are known to to incorporate unnatural amino acids . With the recent devel- 45 those of ordinary skill in the art ( see, the references above , opment of enzymatic ligation and native chemical ligation of and Debinski , et al . ( 1993 ) J. Biol. Chem ., 268 : 14065 peptide fragments , it is possible to make larger proteins . See , 14070 ; Kreitman and Pastan ( 1993 ) Bioconjug . Chem . , 4 : e . g ., P . E . Dawson and S . B . H . Kent, Annu . Rev. Biochem , 581 - 585 ; and Buchner , et al. , (1992 ) Anal. Biochem ., 205 : 69 : 923 ( 2000 ) . Chemical peptide ligation and native chemi- 263 - 270 ) . Debinski, et al. , for example , describe the dena cal ligation are described in U . S . Pat . No. 6 , 184 ,344 , U . S . 50 turation and reduction of inclusion body proteins in guani Patent Publication No . 2004 /0138412 , U . S . Patent Publica - dine -DTE . The proteins can be refolded in a redox buffer tion No. 2003 /0208046 , WO 02 /098902 , and WO containing , including but not limited to , oxidized gluta 03 /042235 , which are incorporated by reference herein . A thione and L -arginine . Refolding reagents can be flowed or general in vitro biosynthetic method in which a suppressor otherwise moved into contact with the one or more poly tRNA chemically acylated with the desired unnatural amino 55 peptide or other expression product, or vice - versa . acid is added to an in vitro extract capable of supporting In the case of prokaryotic production of relaxin polypep protein biosynthesis , has been used to site -specifically incor - tide , the relaxin polypeptide thus produced may be mis porate over 100 unnatural amino acids into a variety of folded and thus lacks or has reduced biological activity . The proteins of virtually any size . See , e . g ., V . W . Cornish , D . bioactivity of the protein may be restored by “ refolding” . In Mendel and P . G . Schultz , Angew . Chem . Int. Ed . Engl. , 60 general, misfolded relaxin polypeptide is refolded by solu 1995, 34 :621 (1995 ); C . J. Noren , S . J. Anthony -Cahill , M . bilizing (where the relaxin polypeptide is also insoluble ) , C . Griffith , P . G . Schultz , A general method for site -specific unfolding and reducing the polypeptide chain using , for incorporation of unnatural amino acids into proteins , Sci- example , one or more chaotropic agents ( e . g . urea and / or ence 244 : 182 - 188 ( 1989) ; and , J . D . Bain , C . G .Glabe , T . A . guanidine ) and a reducing agent capable of reducing disul Dix , A . R . Chamberlin , E . S . Diala , Biosynthetic site -specific 65 fide bonds ( e . g . dithiothreitol, DTT or 2 -mercaptoethanol , incorporation of a non -natural amino acid into a polypep - 2 -ME ) . At a moderate concentration of chaotrope , an oxi tide, J. Am . Chem . Soc, 111: 8013 - 8014 ( 1989 ). A broad dizing agent is then added (e . g ., oxygen , cystine or cysta US 9 , 962 , 450 B2 121 122 mine ), which allows the reformation of disulfide bonds. and SUPERFRAC® Fraction Collector (Amersham Biosci Relaxin polypeptide may be refolded using standard meth ences , Piscataway , N . J . ) . Mixers are also available to form ods known in the art , such as those described in U . S . Pat . pH and linear concentration gradients . Commercially avail Nos. 4 ,511 ,502 , 4 ,511 , 503 , and 4 , 512 ,922 , which are incor - able mixers include Gradient Mixer GM - 1 and In - Line porated by reference herein . The relaxin polypeptide may 5 Mixers ( Amersham Biosciences, Piscataway, N V . J . ). . also be cofolded with other proteins to form heterodimers or The chromatographic process may be monitored using heteromultimers . any commercially available monitor. Such monitors may be After refolding , the relaxin may be further purified . Puri - used to gather information like UV, pH , and conductivity . fication of relaxin may be accomplished using a variety of Examples of detectors include Monitor UV - 1 , UVICORD techniques known to those of ordinary skill in the art , 10 S II, Monitor UV - M II , Monitor UV - 900 , Monitor UPC - 900 , including hydrophobic interaction chromatography, size Monitor pH / C - 900 , and Conductivity Monitor (Amersham exclusion chromatography , ion exchange chromatography, Biosciences, Piscataway, N . J . ) . Indeed , entire systems are reverse -phase high performance liquid chromatography, commercially available including the various AKTA? sys affinity chromatography, and the like or any combination tems from Amersham Biosciences (Piscataway , N . J. ). thereof. Additional purification may also include a step of 15 In one embodiment of the present invention , for example , drying or precipitation of the purified protein . the relaxin polypeptide may be reduced and denatured by After purification , relaxin may be exchanged into differ - first denaturing the resultant purified relaxin polypeptide in ent buffers and / or concentrated by any of a variety of urea , followed by dilution into TRIS buffer containing a methods known to the art , including , but not limited to , reducing agent (such as DTT ) at a suitable pH . In another diafiltration and dialysis. Relaxin that is provided as a single 20 embodiment, the relaxin polypeptide is denatured in urea in purified protein may be subject to aggregation and precipi - a concentration range of between about 2 M to about 9 M , tation . followed by dilution in TRIS buffer at a pH in the range of The purified relaxin may be at least 90 % pure (as mea about 5 . 0 to about 8 . 0 . The refoldingmixture of this embodi sured by reverse phase high performance liquid chromatog - ment may then be incubated . In one embodiment, the raphy, RP -HPLC , or sodium dodecyl sulfate - polyacrylamide 25 refolding mixture is incubated at room temperature for four gel electrophoresis, SDS- PAGE) or at least 95 % pure , or at to twenty -four hours . The reduced and denatured relaxin least 98 % pure , or at least 99 % or greater pure. Regardless polypeptide mixture may then be further isolated or purified . of the exact numerical value of the purity of the relaxin , the As stated herein , the pH of the first relaxin polypeptide relaxin is sufficiently pure for use as a pharmaceutical mixture may be adjusted prior to performing any subsequent product or for further processing , such as conjugation with 30 isolation steps . In addition , the first relaxin polypeptide a water soluble polymer such as PEG . mixture or any subsequent mixture thereof may be concen Certain relaxin molecules may be used as therapeutic trated using techniques known in the art. Moreover , the agents in the absence of other active ingredients or proteins elution buffer comprising the first relaxin polypeptide mix ( other than excipients , carriers, and stabilizers, serum albu - ture or any subsequent mixture thereof may be exchanged min and the like ), or they may be complexed with another 35 for a buffer suitable for the next isolation step using tech protein or a polymer. niques known to those of ordinary skill in the art . General Purification Methods Ion Exchange Chromatography Any one of a variety of isolation steps may be performed In one embodiment, and as an optional , additional step , on the cell lysate , extract, culture medium , inclusion bodies, ion exchange chromatography may be performed on the first periplasmic space of the host cells , cytoplasm of the host 40 relaxin polypeptide mixture . See generally ION cells , or other material, comprising relaxin polypeptide or on EXCHANGE CHROMATOGRAPHY : PRINCIPLES AND any relaxin polypeptide mixtures resulting from any isola METHODS (Cat . No . 18 -1114 - 21, Amersham Biosciences tion steps including , but not limited to , affinity chromatog (Piscataway , N . J. ) ) . Commercially available ion exchange raphy, ion exchange chromatography, hydrophobic interac columns include HITRAP® , HIPREP® , and HILOAD® tion chromatography, gel filtration chromatography, high 45 Columns (Amersham Biosciences , Piscataway , N . J . ) . Such performance liquid chromatography (“ HPLC ” ) , reversed columns utilize strong anion exchangers such as Q SEP phase -HPLC (“ RP -HPLC ” ) , expanded bed adsorption , or HAROSE® Fast Flow , OSEPHAROSE® High Perfor any combination and /or repetition thereof and in any appro mance , and Q SEPHAROSE® XL , strong cation exchangers priate order. such as SP SEPHAROSE High Performance , SP SEPHAR Equipment and other necessary materials used in perform - 50 OSE® Fast Flow , and SP SEPHAROSE® XL ; weak anion ing the techniques described herein are commercially avail - exchangers such as DEAE SEPHAROSE® Fast Flow ; and able . Pumps, fraction collectors , monitors , recorders , and weak cation exchangers such as CM SEPHAROSE® Fast entire systems are available from , for example , Applied Flow (Amersham Biosciences , Piscataway , N . J . ) . Anion or Biosystems (Foster City, Calif. ) , BioRad Laboratories, Inc . cation exchange column chromatography may be performed (Hercules , Calif . ) , and Amersham Biosciences , Inc . ( Piscat - 55 on the relaxin polypeptide at any stage of the purification away , N . J . ). Chromatographic materials including , but not process to isolate substantially purified relaxin polypeptide. limited to , exchange matrix materials , media , and buffers are The cation exchange chromatography step may be per also available from such companies. formed using any suitable cation exchange matrix . Useful Equilibration , and other steps in the column chromatog - cation exchange matrices include , but are not limited to , raphy processes described herein such as washing and 60 fibrous , porous , non -porous , microgranular, beaded , or elution , may be more rapidly accomplished using special- cross - linked cation exchange matrix materials . Such cation ized equipment such as a pump. Commercially available exchange matrix materials include , but are not limited to , pumps include, but are not limited to, HILOAD® Pump cellulose , agarose , dextran , polyacrylate , polyvinyl, poly P -50 , Peristaltic Pump P -1 , Pump P - 901, and Pump P - 903 styrene, silica , polyether , or composites of any of the fore (Amersham Biosciences, Piscataway , N . J. ) . 65 going . Examples of fraction collectors include RediFrac Fraction The cation exchange matrix may be any suitable cation Collector , FRAC - 100 and FRAC - 200 Fraction Collectors , exchanger including strong and weak cation exchangers . US 9 , 962 , 450 B2 123 124 Strong cation exchangers may remain ionized over a wide to , at least about C3 to at least about C30 , at least about C3 pH range and thus, may be capable of binding relaxin over to at least about C20 , or at least about C3 to at least about a wide pH range . Weak cation exchangers , however , may C18 , resins may be used . Alternatively , a polymeric resin lose ionization as a function of pH . For example , a weak may be used . For example , TosoHaas Amberchrome cation exchanger may lose charge when the pH drops below 5 CG1000sd resin may be used , which is a styrene polymer about pH 4 or pH 5 . Suitable strong cation exchangers resin . Cyano or polymeric resins with a wide variety of alkyl include, but are not limited to , charged functional groups chain lengths may also be used . Furthermore, the RP - HPLC such as sulfopropyl (SP ) , methyl sulfonate ( S ) , or sulfoethyl column may be washed with a solvent such as ethanol . The ( SE ) . The cation exchange matrix may be a strong cation Source RP column is another example of a RP -HPLC exchanger, preferably having a relaxin binding pH range of 10 column . about 2 . 5 to about 6 . 0 . Alternatively , the strong cation A suitable elution buffer containing an ion pairing agent exchanger may have a relaxin binding pH range of about pH and an organic modifier such as methanol, isopropanol, 2 . 5 to about pH 5 . 5 . The cation exchange matrix may be a tetrahydrofuran , acetonitrile or ethanol, may be used to elute strong cation exchanger having a relaxin binding pH of the relaxin polypeptide from the RP -HPLC column. The about 3 . 0 . Alternatively , the cation exchange matrix may be 15 most commonly used ion pairing agents include , but are not a strong cation exchanger, preferably having a relaxin bind - limited to , acetic acid , formic acid , perchloric acid , phos ing pH range of about 6 . 0 to about 8 .0 . The cation exchange phoric acid , trifluoroacetic acid , heptafluorobutyric acid , matrix may be a strong cation exchanger preferably having triethylamine , tetramethylammonium , tetrabutylammonium , a relaxin binding pH range of about 8 . 0 to about 12 . 5 . and triethylammonium acetate . Elution may be performed Alternatively , the strong cation exchanger may have a 20 using one or more gradients or isocratic conditions , with relaxin binding pH range of about pH 8 . 0 to about pH 12 . 0 . gradient conditions preferred to reduce the separation time Prior to loading the relaxin , the cation exchange matrix and to decrease peak width . Another method involves the may be equilibrated , for example , using several column use of two gradients with different solvent concentration volumes of a dilute , weak acid , e . g . , four column volumes of ranges . Examples of suitable elution buffers for use herein 20 mM acetic acid , pH 3 . Following equilibration , the 25 may include , but are not limited to , ammonium acetate and relaxin may be added and the column may be washed one to acetonitrile solutions . several times , prior to elution of substantially purified Hydrophobic Interaction Chromatography Purification relaxin , also using a weak acid solution such as a weak acetic Techniques acid or phosphoric acid solution . For example , approxi - Hydrophobic interaction chromatography (HIC ) may be mately 2 - 4 column volumes of 20 mM acetic acid , pH 3 , 30 performed on the relaxin polypeptide . See generally may be used to wash the column . Additional washes using , HYDROPHOBIC INTERACTION CHROMATOGRA e. g ., 2 - 4 column volumes of 0 .05 M sodium acetate , pH 5 .5 , PHY HANDBOOK : PRINCIPLES AND METHODS (Cat . or 0 .05 M sodium acetate mixed with 0 . 1 M sodium chlo No. 18 - 1020 - 90 , Amersham Biosciences (Piscataway , N . J . ) ride , pH 5 . 5 , may also be used . Alternatively , using methods which is incorporated by reference herein . Suitable HIC known in the art , the cation exchange matrix may be 35 matrices may include , but are not limited to , alkyl- or equilibrated using several column volumes of a dilute , weak aryl- substituted matrices, such as butyl- , hexyl - , octyl - or base . phenyl- substituted matrices including agarose , cross- linked Alternatively , substantially purified relaxin may be eluted agarose , sepharose, cellulose , silica , dextran , polystyrene , by contacting the cation exchanger matrix with a buffer poly (methacrylate ) matrices , and mixed mode resins , having a sufficiently low pH or ionic strength to displace the 40 including but not limited to , a polyethyleneamine resin or a relaxin from the matrix . The pH of the elution buffer may butyl- or phenyl- substituted poly (methacrylate ) matrix . range from about PH 2 . 5 to about pH 6 . 0 . More specifically , Commercially available sources for hydrophobic interaction the pH of the elution buffer may range from about PH 2 . 5 to column chromatography include, but are not limited to , about pH 5 . 5 , about pH 2 . 5 to about pH 5 .0 . The elution HITRAP® , HIPREP® , and HILOAD® columns (Amer buffer may have a pH of about 3 . 0 . In addition , the quantity 45 sham Biosciences, Piscataway, N . J . ) . of elution buffer may vary widely and will generally be in Briefly , prior to loading , the HIC column may be equili the range of about 2 to about 10 column volumes. brated using standard buffers known to those of ordinary Following adsorption of the relaxin polypeptide to the skill in the art , such as an acetic acid /sodium chloride cation exchanger matrix , substantially purified relaxin poly solution or HEPES containing ammonium sulfate . Ammo peptide may be eluted by contacting the matrix with a buffer 50 nium sulfate may be used as the buffer for loading the HIC having a sufficiently high pH or ionic strength to displace the column . After loading the relaxin polypeptide , the column relaxin polypeptide from the matrix . Suitable buffers for use may then washed using standard buffers and conditions to in high pH elution of substantially purified relaxin polypep - remove unwanted materials but retaining the relaxin poly tide may include , but not limited to , citrate , phosphate , peptide on the HIC column . The relaxin polypeptide may be formate , acetate , HEPES , and MES buffers ranging in con - 55 eluted with about 3 to about 10 column volumes of a centration from at least about 5 mM to at least about 100 standard buffer, such as a HEPES buffer containing EDTA mM . and lower ammonium sulfate concentration than the equili Reverse - Phase Chromatography brating buffer, or an acetic acid /sodium chloride buffer, RP - HPLC may be performed to purify proteins following among others . A decreasing linear salt gradient using , for suitable protocols that are known to those of ordinary skill 60 example , a gradient of potassium phosphate , may also be in the art . See , e. g ., Pearson et al. , ANAL BIOCHEM . used to elute the relaxin molecules . The eluant may then be ( 1982 ) 124 :217 - 230 ( 1982 ) ; Rivier et al. , J. CHROM . ( 1983 ) concentrated , for example , by filtration such as diafiltration 268: 112 - 119 ; Kunitani et al. , J . CHROM . ( 1986 ) 359: 391 - or ultrafiltration . Diafiltration may be utilized to remove the 402. RP -HPLC may be performed on the relaxin polypep - salt used to elute the relaxin polypeptide . tide to isolate substantially purified relaxin polypeptide . In 65 Other Purification Techniques this regard , silica derivatized resins with alkyl functional- Yet another isolation step using, for example, gel filtration ities with a wide variety of lengths, including , but not limited (GEL FILTRATION : PRINCIPLES AND METHODS (Cat .