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(12) Patent Application Publication (10) Pub. No.: US 2006/0234346A1 Retallack Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2006/0234346A1 Retallack Et Al US 20060234346A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0234346A1 Retallack et al. (43) Pub. Date: Oct. 19, 2006 (54) EXPRESSION OF MAMMALIAN PROTEINS (60) Provisional application No. 60/564,798, filed on Apr. N PSEUDOMONAS FLUORESCENS 22, 2004. Provisional application No. 60/537,148, filed on Jan. 16, 2004. Provisional application No. 60/417,124, filed on Oct. 8, 2002. (76) Inventors: Diane M. Retallack, Poway, CA (US); Charles H. Squires, Poway, CA (US); Publication Classification David C. Watkins, East Greenwich, RI (51) Int. Cl. (US); Stacey L. Lee, San Diego, CA CI2P 2/06 (2006.01) (US); Frank H. Gaertner, San Diego, C07K I4/47 (2006.01) CA (US); Robert Shutter, Poway, CA CI2N I/2 (2006.01) (US) CI2N 15/74 (2006.01) C7H 2L/04 (2006.01) Correspondence Address: (52) U.S. Cl. ................... 435/69.1; 435/471; 435/252.34; KING & SPALDING LLP 530/350, 536/23.1 118O PEACHTREE STREET ATLANTA, GA 30309 (US) (57) ABSTRACT (21) Appl. No.: 11/400,840 The invention is a process for improved production of a recombinant mammalian protein by expression in a (22) Filed: Apr. 7, 2006 Pseudomonad, particularly in a Pseudomonas fluorescens organism. The process improves production of mammalian Related U.S. Application Data proteins, particularly human or human-derived proteins, over known expression systems such as E. coli in compa (63) Continuation of application No. 11/038,901, filed on rable circumstances Processes for improved production of Jan. 18, 2005, and which is a continuation-in-part of isolated mammalian, particularly human, proteins are pro application No. 10/681,540, filed on Oct. 7, 2003. vided. Patent Application Publication Oct. 19, 2006 Sheet 1 of 11 US 2006/0234346 A1 Amount of Virus or IFN Added FIGURE 1 Patent Application Publication Oct. 19, 2006 Sheet 2 of 11 US 2006/0234346 A1 25ng 5Ong //§ |- 2/§. 100ng FIGURE 2 Patent Application Publication Oct. 19, 2006 Sheet 3 of 11 US 2006/0234346A1 A. MFPTIPLSRPFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESI PTPSNREETOQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDL EEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVE TFLRIVQCRSVEGSCGF (SEQ ID NO: 16) B. rid Kan Smal 412-001.hgh asbp \ - Bately " . ceding sage Patent Application Publication Oct. 19, 2006 Sheet 4 of 11 US 2006/0234346 A1 O Sol IO ins 24 Sol 24 ins 48 Sol 48 ins s i : FIGURE 4 Patent Application Publication Oct. 19, 2006 Sheet 5 of 11 US 2006/0234346 A1 48 24 O % y-iFN per fraction 5 95 R 94 n 1 n? FIGURES Patent Application Publication Oct. 19, 2006 Sheet 6 of 11 US 2006/0234346 A1 EcoRI (1429s No I (408) OV ra MobAB RapB. A No1 (11284)-77 NoI (11186). Nool (10543) HindIII (3799) Rp AN A SN4. Pat (3811) NeoI (9385)Y W. BamH. Nao (8808) Y Vi \\Banha\\ ac (3819) Rap C Y.N. W \V Spel (4082) Psil (7974) snai (4276 Tras is series . hoI (7721) - A W . XhoI (511) EcoRI (748) f. w aia EcoRI (7276 Pet (6641) FIGURE 6 Patent Application Publication Oct. 19, 2006 Sheet 7 of 11 US 2006/0234346 A1 agagaactagtaaaaaggagaaatcCATGCAGGGCCAATTTTTTAGA 1. tetcttgatcattttitectetttagg.TACGTCCCGGTTAAAAAATCT--------- a -a -u- - -a- sh as as a asars -ansas pass spous 47 M to G o F. F R 48 ---------GAAATAGAAAACTTAAAGGAGTATTTTAATGCAAGTAGCCCAGATGTAGCTAAGGGTGGG as ance as so as a -a as oos uses spanse o-a--- masas as a so as +---------- lo7 CT TTATCTTTTGAATTTCCTCATAAAATTACGTTCATCGGGTCTACATCGATTcccAccC E I E N L, K E - Y F N A S S P D v A K G G ccTCTCTTCTCAGAAATTTTGAAGAATTGGAAAGATGAAAGTGACAAAAAAATTATTCAG los GGAGAGAAGAGTCTTTAAAACTTCTTAACCTTTCTACTTTCACTGTTTTTTTAATAAGTC--------- east an as a re-ass mann - as a was me seam serie-aa as as a season -- - - - s - 167 P L F S E I L. K. N. W. K. D. E S D K K I I Q AGCCAAATTGTCTCCTTCTACTTCAAACTCTTTGAAAACCTCAAAGATAACCAGGTCATTal 68 TCGGTTTAACAGAGGAAGATGAAGTTTGAGAAACTTTTGGAGTTTCTATTGGTccAGTAA-------------------- -- - - - - - - manus +-------------------- as as a-- - - - -a- + 227 is o I v S F Y F K L E E N L. K D N Q. v I 228 cAAAGGAGCATGGATATCATCAAGCAAGACATGTTTCAGAAGTTCTTGAATGGCAGCTCT----------------------- -an-a-swi-a--- a--- - - -a -a-a-a---- mosaic a as a son on as as v - 287 GTTTCCTCGTACCTATAGTAGTTCGTTCTGTACAAAGTCTTCAAGAACTTACCGTCGAGA Q R S M D I I K Q D M F. Q. K F L N G S S 288 GAGAAACTGGAGGACTTCAAAAAGCTGATTCAAATTCCGGTGGATGATCTGCAGATCCAG--------- as as a -a -um as a - - - - - - - - - -- or as a - - - as as as so man as as - - - - - - - -- 347 circTTTGACCTCCTGAAGTTTTTCGACTAAGTTTAAGGCCACCTACTAGACGTCTAGGTC E K L E D F : K K L I Q I P v D D L Q I a cGCAAAGCCATAAATGAACTCATCAAAGTGATGAATGACCTGTCACCAAAATCTAACCTC 34 B GCGTTTCGGTATTTACTTGAGTAGTTTCACTACTTACTGGACAGTGGTTTTAGATTGGAG--------- a t- a s1 - - ea - as as a as a sh no as as a sa- - -- a---a -- - - - as - us pause --- + 4O7 R. K. A I N E L I K v M N D L S P K S N L. AGAAAGCGGAAGAGAAGTCAGAATCTCTTTCGAGGCCGGAGAGCATCAACGtaatgactegagtetct 408 TCTTTCGCCTTCTCTTCAGTCTTAGAGAAAGCTCCGGCCTCTCGTAGTTGCattactgageteagaga--------- on a - as as - - - - - - - - - - - - so as as as as son an -- a sa- - -- - - - - - - - - - - - - - - - - - -ar as - 45 R K R K R S o N. L. F R G R R A S T * (SEQ ID NO: 17) FIGURE 7 Patent Application Publication Oct. 19, 2006 Sheet 8 of 11 US 2006/0234346 A1 atg eae to aaa Cut Cto estig gcg gea atg aet t t gte get gct GGC 83 Met Lys Let Lys Arg Lea bet Ala Ala at Tarr Phet Val Ala Ala Gly l s l S gtt gog ace occ aec scg gtg gee gee cag gtg cag cts sag gag teg 96 val. Ala Thr. Ala Asn Alaval Ala Ala Glin vali Gln Leg Gla Glu ser 2 2S - O age cea age cts otg aag cct tog gag ace cts tec cte see tge act 84 Gay Pro Gay Leo Val lys Pro ser Gluhr leaser leg tar Cys Tar S - O r. s gte tet gigt gigt tec etc agt agt tat cac tog age to ate egg cag 92 Val Ser Gly Gly Ser le Ser Ser Tyr Bis Trap Ser Tzip lo Arg Gla SO SS SO Cee Cea ogg aag Gga etg gag tog act gigg tat atc tat tac agt gigg 20 Pro Pro Cly Ys Cly leu Gu Tap Ie Gly yar le tyr Tyr Ser Gly 6S O s 90 ege acc aeC tac aac CCC tec Cte aag as Cgan gte acc atta tect gta 23 Ser Thr Asa Tyr Aan Pro Ser Leu Lys Asa Arg Val Tar le Sar Wall 8S 90 95 " 'gac ecs tec eag sac cag tte tec citg anectg agg tet' stg ace get 6 Asp Thr Ser Yo Man Glin Phe Sar Rea Asa Luna Arg serval tyr Ala 100. SOS O .. s gCa gar DCG GCC G to tat tae tgt geg" ega giga acg. tat gigc cca gec - Ala Aap Thir Ala Val Tyr tyr'Cys Ala Arg Gly. Thr Tyr Gly. Pro Ala lis. 320 2S gga gat get titt gat ate tog gigg caa gigg acc acg gue acc gte too 492 Gly Asp Ala Phe Asp Ile rap Gly Gla Gly hr hr Val Thr Val ser 5 ldo agt Ggt oga grgC grt tea gigc giga gigt gige age gge gigt gige giga tog 480 . Ser Gly Gly Gly Gly Ser Gly Guy Gly Gly Ser Gly Gly Gly Gly ser lds SB SS lso gac atc cag atg ace cag tet Cet tec ace etg tet gea tet att. ova S28 As Elr Gla Met Thr Gla Ser Pro Sar Thr Lou Ser Ala Sar la Gly 6s 70 75 gae aga gte ace atc acc tyc cgg gcc agt gag gigt att tat Cae tag SS Asp Arg Val Thr Ile Thr Cys Arg Ala Sar Glu cly rle Tyr is Trip 85 90 . ttg gcc tog tat cag cag aag cea ogg aaa gee ect aaa cte eto atc 628 Leu Ala Trp Tyr Gin Gla Lys Pro Gay Lya Ala Pro Lys at Let le SS 200 20S tat aag gcc tet agt tta gec agt gigg gcc cca tea agg tte age ggc 52 Tyr Lys Ala Ser Ser Leu Alasar Gly Ala Pro Ser Arg rise see Gly 20 2S .220 agt oga tet gigg aea gat tte act ete ace etc age age cts ceg Cet 720 sar Gay Sar Gly Tar Aap Phe Thr Leu. The Elle Ser Ser at cle reo 225 20 2S 280 get gat ttt gea act tat tae tge can caa tat agt aat Cat Cog Ctic 768 Asp Asp Phe Ala Thr Tyr Tyr cys Glin Glin Tyr Ser Asa Tyr 9 eu 265 2SO 2SS act tte gigC gga gigg ace aag ctg gag atc. aaa Cigt GCG gee goa Cat . 86 gar Phe Gly Gly Gly. The Lys Reu Gu Ile Lys Arg Ala Ale Ala Bie 250 26S 270 case Cat Cat COC Cat taa (SEQ ID NO: 18) e as His is is is 275 FIGURE 8 Patent Application Publication Oct. 19, 2006 Sheet 9 of 11 US 2006/0234346 A1 set Lys Leu Lys Arg Leu et Ala Ala Met Thr Fhe val Ala Ala Gly Val Ala Thr Ala Asn Ala Val Ala Ala Glin Val Glin Leu Glin Glu Ser 20 25 30 Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr 35 60 45 . Val Ser Gly Gly Ser IIe Ser Ser Tyr His Trip Ser Trip Ile Arg Glin SO - " .. 55 60 Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly 65 70 75. - 8O ser Thr Asn TYr Asn Pro Ser Leu Lys Asn Arg Val Thr Ilie Ser Val - 85 w 90 9S Asp Thr Ser Lys Asn. Glin Phe Ser Leu Asn Leu Arg Ser Val Thir Ala OO O5 O Ala Asp Thr Ala Val TYr Yr Cys Ala Arg Gly thr Tyr 5 .
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