USOO8906636B2
(12) United States Patent (10) Patent No.: US 8,906,636 B2 Retallack et al. (45) Date of Patent: Dec. 9, 2014
(54) HIGH LEVEL EXPRESSION OF 5,169,760 A 12, 1992 Wilcox RECOMBINANT TOXIN PROTEINS 5,281,532 A 1/1994 Rammler et al. 5,389,540 A 2f1995 Makoff et al. 5,427,788 A 6/1995 Rappuoli et al. (71) Applicant: Pfenex Inc., San Diego, CA (US) 5,443,966 A 8, 1995 Fairweather et al. 5,571,694 A 11/1996 Makoff et al. (72) Inventors: Diane M. Retallack, Poway, CA (US); 5,614,382 A 3, 1997 Metcalf Lawrence Chew, San Diego, CA (US) 5,773,600 A 6/1998 Burnette, III 5,785,971 A 7/1998 Rappuoli et al. 5,792.458 A 8, 1998 Johnson et al. (73) Assignee: Pfenex Inc., San Diego, CA (US) 5,834,246 A 11/1998 Holmgren et al. 5,919,463 A 7/1999 Thomas, Jr. et al. (*) Notice: Subject to any disclaimer, the term of this 5,935,580 A 8, 1999 Ladant et al. patent is extended or adjusted under 35 6,010,871 A 1/2000 Takahara et al. U.S.C. 154(b) by 0 days. 6,043,057 A 3/2000 Holmgren et al. 6,140,082 A 10/2000 Loosmore et al. 6,733,760 B1 5, 2004 Wilkins et al. (21) Appl. No.: 13/952,484 6,939,548 B2 9, 2005 Wilkins et al. 7,169,399 B2 1/2007 Roberts (22) Filed: Jul. 26, 2013 7,226,597 B2 6/2007 Ballard et al. 7,232,671 B2 6/2007 Cieplak (65) Prior Publication Data 7,273,728 B2 9, 2007 Wolfe et al. 7.427.404 B1 9, 2008 Pizza et al. US 2014/OO51093 A1 Feb. 20, 2014 7,575,891 B2 8, 2009 Wolfe et al. 7,618,799 B2 11/2009 Coleman et al. Related U.S. Application Data 7,666.436 B1 2/2010 Pizza et al. 7,985,564 B2 7/2011 Retallack et al. (60) Division of application No. 13/073.955, and a 8,288,127 B2 10/2012 Schneider et al. continuation-in-part of application No. PCT/ 8,530,171 B2 9/2013 Retallack et al. US2010/030573, filed on Apr. 9, 2010, now Pat. No. (Continued) 8,530,171. (60) Provisional application No. 61/325,235, filed on Apr. FOREIGN PATENT DOCUMENTS 16, 2010, provisional application No. 61/319,152, EP 0207459 1, 1978 filed on Mar. 30, 2010. EP O478602 1, 1996 WO WO-90-09444 8, 1990 WO WO-90-15871 12/1990 (51) Int. Cl. WO WO-97-02836 1, 1997 CI2N 9/10 (2006.01) WO WO-2005-000346 1, 2005 CI2P 2L/00 (2006.01) WO WO-2005-0521.51 6, 2005 GOIN 33/573 (2006.01) WO WO-2005-056773 6, 2005 CI2N 15/78 (2006.01) WO WO-2005-0699.13 8, 2005 CI2P 21/02 (2006.01) WO WO-2005-089093 9, 2005 C07K (4/34 (2006.01) (Continued) (52) U.S. Cl. CPC ...... CI2N 9/1051 (2013.01); C12N 15/78 OTHER PUBLICATIONS (2013.01); CI2P21/00 (2013.01); C07K Yang et al., BMC Microbiology 8, article 192 (2008).* 2319/036 (2013.01); C12P21/02 (2013.01); Ellingsworth, L., Pseudomonas fluorescens: Expression System for C07K 14/34 (2013.01); G0IN33/573 (2013.01) Producing Recombinant Vaccines and Adjuvants (2006).* USPC ...... 435/7.4; 435/15; 435/23: 435/69.3: Allured et al., Structure of exotoxin A of Pseudomonas aeruginosa at 435/193:435/471 3.0-Angrstom resolution, PNAS USA 83:1320-1324 (1986). (58) Field of Classification Search CPC ...... C12N 15/78; C12N 9/1051; C12P 21/00; (Continued) GO1 N 33/573 USPC ...... 435/7.4, 15, 23, 69.3, 193, 471 Primary Examiner — Chih-Min Kam See application file for complete search history. (74) Attorney, Agent, or Firm — Wilson Sonsini Goodrich & Rosati (56) References Cited U.S. PATENT DOCUMENTS (57) ABSTRACT 4,551,433 A 11, 1985 DeBoer The present invention relates to the field of recombinant toxin 4,695.455 A 9, 1987 Barnes et al. protein production in bacterial hosts. In particular, the present 4,709,017 A 11, 1987 Collier invention relates to production processes for obtaining high 4,755.465 A 7/1988 Gray et al. 4,830,962 A 5, 1989 Gelfand et al. levels of a recombinant CRM197, Diphtheria Toxin, Pertussis 4,861,595 A 8, 1989 Barnes et al. Toxin, Tetanus Toxoid Fragment C. Cholera Toxin B, Cholera 4,892,827 A 1/1990 Pastan et al. holotoxin, and Pseudomonas EXotoxin A, from a bacterial 4,925,792 A 5/1990 Rappuoli host. 5,055,294 A 10/1991 Gilroy 5,085,862 A 2f1992 Klein et al. 5,128,130 A 7/1992 Gilroy et al. 18 Claims, 59 Drawing Sheets US 8,906,636 B2 Page 2
(56) References Cited Frishman et al., Starts of Bacterial Genes: Estimating the Reliability of Computer Predictions, Gene 234(20): 257-265 (1999). U.S. PATENT DOCUMENTS Giannini et al., The amino acid sequence of two non-toxic mutants of diphtheria toxin: CRM45 and CRM 197, Nucl Acids Res 8,603,824 B2 12/2013 Ramseier et al. 2003,0224009 A1 12/2003 Terry et al. 12(10):4063-4069 (1984). 2006, OOO8877 A1 1/2006 Retallack et al. Greenfield et al., Nucleotide sequence of the structural gene for 2006,0040352 A1 2/2006 Retallack et al. diphtheria toxin carried by corynebacteriophage B, PNAS USA 2006, O110747 A1 5, 2006 Ramseier et al. 80:6853-6857 (1983). 2006/0234346 A1 10, 2006 Retallack et al. Gurkin and Ellar, Recombinant production of bacterial toxins and 2006, O246036 A1 11/2006 Francis et al. their derivatives in the methylotrophic yeast Pichia pastoris, Micro 2007/0292918 A1 12/2007 Stelman et al. 2008/O193974 A1 8/2008 Coleman et al. bial Cell Factories 4:33 (2005). 2008/0269070 A1 10/2008 Ramseier et al. Haemophilus b Conjugate Vaccine (Diphtheria CRM197 Protein 2009,0081184 A1 3/2009 Margolin et al. Conjugate (HibTiter) Package Insert, 17 pages dated Jan. 2007. 2009/0325230 A1 12/2009 Schneider et al. Harakunietal. Heteropentameric Cholera Toxin B Subunit Chimeric 2010.0048864 A1 2/2010 Coleman et al. Molecules Genetically Fused to a Vaccine Antigen Induce Systemic 2010.0137162 A1 6, 2010 Retallack and Mucosal Immune Responses: a Potential New Strategy to Target 2011/0287443 A1 11/2011 Retallack et al. Recombinant Vaccine Antigens to Mucosal Immune Systems, Infec 2012,02896.88 A1 11/2012 Blais et al. tion and Immunity 73(9): 5654-5665 (2005). 2014/0051093 A1 2/2014 Retallack et al. Ikehata et al., Primary structure of nitrile hydratase deduced from the nucleotide sequence of a Rhodococcus species and its expression in FOREIGN PATENT DOCUMENTS Escherichia coli, EurJ Biochem 181(3):563-570 (1989). WO WO-2006-014899 2, 2006 Jank and Aktories, Structure and mode of action of clostridial WO WO-2007-146139 12/2007 glucosylating toxins: the ABCD model, Trends in Microbiol. WO WO-2008-094986 8, 2008 16(5):222-229 (2008). WO WO-2008-134461 11, 2008 Kaslow et al., Structure-Activity Analysis of the Activation of Pertus WO WO-2010-008764 1, 2010 sis Toxin, Biochem 26(1): 123-127 (1987). WO WO-2011-042516 4/2011 Kinket al. Antibodies to Recombinant Clostridium dificile Toxins A and B Arean Effective Treatment and Prevent Relapse of C. difficile OTHER PUBLICATIONS Associates Disease in a Hamster Model of Infection, Infection and Immunity, 66(5):2018-2025 (May 1998). Anderson et al., Safety and Immunogenicity of Meningococcal A and Kulich et al., Expression of Recombinant Exoenzyme S of C Polysaccharide Conjugate Vaccine in Adults, Infection and Immu Pseudomonas aeruginosa, Infection and Immunity 63(1): 1-8 nity 62(8):3391-3395 (1994). (1995). AU App No. 2010201410 Examination Report dated Jun. 6, 2014. Lee et al., Characterization of a Cloned Temperature-Sensitive Con Barth et al., Binary Bacterial Toxins: Biochemistry, Biology, and struct of the Diphtheria Toxin A Domain, Biochem 44(7):2555-2565 Applications of Common Clostridium and Bacillus Proteins, (2005) (Abstract). Microbiol Mol Biol Rev 68(3):373-402 (2004). Linet al., The Efficacy of a Salmonella typhi ViConjugate Vaccine in Bergey’s Manual of Determinative Bacteriology, R.E. Buchanan and Two-to-Five Year-Old Children, New England J Med 344(17): 1263 1269 (2001). N.E. Gibbons eds., pp. 217-289, 8th ed., The Williams & Wilkins Co., Lukac et al., Toxoid of Pseudomonas aeruginosa Exotoxin A Gen Baltimore, MD, 1974. erated by Deletion of an Active-Site Residue, Infection and Immunity Bishai et al., High-Level Expression of a Proteolytically Sensitive 56(12):3095-3098 (1988). Diphtheria Toxin Fragment in Escherichia coli, J Bacteriology Maunsell et al., Complex regulation of AprA metalloprotease in 169(11):5140-5151 (1987). Pseudomonas fluorescens M114: evidence for the involvement of Burnette et al., Properties of Pertussis Toxin B Oligomer Assembled iron, the ECF sigma factor, PbrA and pseudobactin M114 In Vitro from Recombinant Polypeptides Produced by Escherichia siderophore, Microbiol 152(Pt 1):29-42 (2006). coli, Infection and Immunity 60(6):2252-2256 (1992). McCoy et al., PAR1 and PAR2 couple to overlapping and distinct sets Carbonetti et al., Proteolytic cleavage of pertussis toxin S1 subunit is of G proteins and linked signaling pathways to differentially regulate not essential for its activity in mammalian cells, BMC Microbiology cell physiology, Molecular Pharmacology Fast Forward. Published 5:7 (2005). on Mar. 9, 2010 as doi:10.1124/mol. 109.062018. CN201080066026.6 Office Action dated May 12, 2014. Mekada and Uchida, Binding Properties of Diphtheria Toxinto Cells CN201080066026.6 Office Action dated Sep. 23, 2013. Are Altered by Mutation in the Fragment A Domain, J Biol Chem Collier, Diphtheria Toxin: Mode of Action and Structure, Bacterio 260(22): 12148-12153 (1985). logical Reviews 39(1):54-85 (1975). MenACWY-CRM Clinical Trial and Product Overview, Novartis CryZ et al., Isolation and characterization of a Pseudomonas Vaccines Jun. 24, 2009—ACIP Meeting Atlanta. aeruginos a mutant producing a nontoxic, immunologically crossac Menveo product insert Feb. 2010. tive toxin A protein, PNAS 77(12):7199-7203 (1980). Miyaji et al., Induction of Neutralizing Antibodies against Diphtheria Davis and Mingioli, Mutants of Escherichia coli requiring Toxin by Priming with Recombinant Mycobacterium bovis BCG methionine or Vitamin B12, Bact 60:17-28 (1950). Expressing CRM197, a Mutant Diphtheria Toxin, Infection and Eisel et al., Tetanus toxin: primary structure, expression in E. coli, Immunity 69(2):869-874 (2001). and homology with botulinum toxins, EMBO J 5(10):2495-2502 Mueller-Dieckmann et al., Structure of mouse ADP-ribosylhydrolase (1986). 3 (mARH3), Acta CrystF64:156-162 (2008). Ellingsworth, Pseudomonas fluorescens: Expression System for Pro Nozoye et al., Production of Ascaris suum AS14 Protein and Its ducing Recombinant Vaccines and Adjuvants, (2006). Fusion Protein with Cholera Toxin B Subunit in Rice Seeds, J. Vet. EP11766437.5 Supplementary European Search Report dated Oct. 9, Med. Sci. 71(7):995-1000 (2009). 2013. NZ602958 Examination Report dated May 17, 2013. Fairweather and Lyness, The complete nucleotide sequence of teta Orr et al., Expression and Immunogenicity of a Mutant Diphtheria nus toxin, Nucl Acids Res 14(19):7809-7812 (1986). Toxin Molecule, CRM197, and Its Fragments in Salmonella typhi Fattom et al., Laboratory and Clinical Evaluation of Conjugate Vac Vaccine Strain CVD 908-htra, Infection and Immunity 67(8):4290 cines Composed of Staphylococcus aureus Type 5 and Type 8 Cap 4294 (1999). Sular Polysaccharides Bound to Pseudomonas aeruginosa Recombi PCT/US 10/30573 International Preliminary Report on Patentability nant Exoprotein A, Infection and Immunity 61(3):1023-1032 (1993). mailed Oct. 11, 2012. US 8,906,636 B2 Page 3
(56) References Cited Schweizer, Vectors to express foreign genes and techniques to moni tor gene expression in Pseudomondas, Curr Op Biotech 12:439-445 OTHER PUBLICATIONS (2001). Sekura et al., Pertussis Toxin. Affinity Purification of a New ADP PCT/US 10/30573 Search Report and Written Opinion mailed May Ribosyltransferase, J Biol Chem 2.58:14647 (1983). 27, 2011. Slater & R. Williams, Molecular Biology and Biotechnology, J. PCT/US2011/030227 International Preliminary Report on Walker & R. Rapley, eds., pp. 125-154. The Royal Society of Chem istry, Cambridge, UK, 2000. Patentabilty mailed Oct. 11, 2012. Stickings et al., Transcutaneous Immunization with Cross-Reacting PCT/US2011/030227 International Search Report and Written Opin Material CRM197 of Diphtheria Toxin Boosts Functional Antibody ion dated Dec. 22, 2011. Levels in Mice Primed Parenterally with Adsorbed Diphtheria Pneumococcal 7-valent Conjugate Vaccine (Diphtheria CRM197 Toxoid Vaccine, Infection and Immunity 76(4): 1766-1773 (2008). Protein) Prevnar Package Insert Wyeth Pharmaceuticals Inc. dated Su et al., Pertussis Toxin Inhibits Induction of Tissue-Specific Oct. 2008. Autoimmune Disease by Disrupting G Protein-Coupled Signals, J Popoffet al., Actin-Specific ADP-Ribosyltransferase Produced by a Immunol 167:250-256 (2001). Clostridium difficule Strain, Infection and Immunity 56(9): 2299 Sullivan et al., Purification and Characterization of Toxins A and B of 2306 (1988). Clostridium difficule, Infection and Immunity 35(3):1032-1040 Price et al., Intranasal Administration of Recombinant Neisseria (1982). gonorrhoeae Transferrin Binding Proteins A and B Conjugated to the Sun et al., Intransal Administration of a Schistosoma mansoni Cholera Toxin B Subunit Induces Systemic and Vaginal Antibodies in Glutathione S-Transferase-Cholera Toxoid Conjugate Vaccine Mice, Infection and Immunity 73(7):3945-3953 (2005). Evokes Antiparasitic and Antipathological Immunity in Mice, J Qian et al., Conugating recombinant proteins to Pseudomonas Immunol 163:1045-1052 (1999). aeruginosa ExoProtein A: a strategy for enhancing immunogenicity Suzek, Baris E., et al., “A Probalistic Method for Identifying Start of malaria vaccine candidates, Vaccine 25(20):3923-3933 (2007). Codons in Bacterial Genomes.” Bioinformatics, 2001, pp. 1123 Retallack, et al., “Transport of heterologous proteins to the 1130, vol. 17, No. 12, Oxford University Press. periplasmic space of Pseudomonas fluorescens using a variety of Townsend et al., Tetanus toxin C fragment conjugated nanoparticles native signal sequences. Biotechnology Letters, Springer Nether for targeted drug delivery to neurons, Biomaterials 28(34):5176 lands, Dordrecht, vol. 29, No. 10, May 31, 2007, pp. 1483-1491. 5184 (2007). Riesenberg et al., High cell density cultivation of Escherichia coli at Tsuge et al., Structural basis of actin recognition and arginine ADP controlled specific growth rate, J Biotechnol 20(1):17-27 (1991). ribosylation by Clostridium perfringens —toxin, PNAS Roberts et al., A Mutant Pertussis Toxin Molecule That Lacks ADP 105(21):7399-7404 (2008). Ribosyltransferase Activity, PT-9K/129G. Is an Effective Mucosal U.S. Appl. No. 13/073.955 Office Action dated Dec. 7, 2012. Adjuvant for Intranasally Delivered Proteins, Infection and Immu U.S. Appl. No. 13/952,484 Office Action dated Apr. 9, 2014. nity 63(6):2100-2108 (1995). Watkins et al., Pertussis Toxin Treatment in Vivo Is Associated with Rodighiero et al., A Cholera Toxin B-subunit Variant That Binds a Decline in G-protein B-Subunits, J Biol Chem 264(7):4186-4194 Ganglioside GM1 but Fails to Induce Toxicity, J Biol Chem (1989). 276(40):36939-36945 (2001). Winram, Development and Preclinical Optimization of a Sakurai et al., Clostridium perfringens Iota-Toxin: Structure and Circumsporozoite Protein (rCSP) Vaccine Candidate Against Function, Toxins 1:208-228 (2009). Malaria Via a Virtual Pharmaceutical Approach, Presented at Pub Sanchez-Romero and V. De Lorenzo, Manual of Industrial lished in BioProcess International 2011, Nov. 3, 2011. Microbiology and Biotechnology, A. Demain & J. Davies, eds., pp. Winram, rCSP Expression Project Utilizing Pfenex Technology 460-474, 1999. rCSP Expression Project Utilizing Pfenex Technology, Presented Schirmer et al., The ADP-ribosylating Mosquitocidal Toxin from at published in Vaccine Production Summit, San Diego, Jun. 14. Bacillus sphaericus, J Biol Chem 277(14): 11941-11948 (2002). 2010. Schmidt and Schmidt, Inhibition of Pertussis Toxin Binding to Model Wozniak et al., Construction and use of a nontoxigenic strain of Receptors by Antipeptide Antibodies Directed at an Antigenic Pseudomonas aeruginosa for the production of recombinant Domain of the S2 Subunit, Infection and Immunity 57(12):3828 exotoxin A., Applied and Environmental Microbiology, vol. 61, No. 3833 (1989). 5, May 1, 1995, pp. 1739-1744. Schneider et al., Auxotropic markers pyrF and proC can replace Yang et al., Expression of recombinant Clostridium difficile toxin A Sntobiotic markers in protein productions plasmids in hig-cell-den and B in Bacillus megaterium, BMC Microbiol 8: 192 (2008). sity Pseudomonas fluorescens fermentation. Biotechnol. Progress Zhou et al Secretory expression of recombinant diphtheria toxin 21(2)(2005): 343-8. mutants in B. subtilis, Journal of Tongji Medical University, vol. 19. Schneider, Enabling Biodefense Countermeasures through Next No. 4, 1999, pp. 253-256. Generation Vaccines, Presented at Published in Phacilitate Vaccine Forum, Jan. 28, 2014. * cited by examiner
U.S. Patent Dec. 9, 2014 Sheet 7 of 59 US 8,906,636 B2
SCHRI[15)I,H.
U.S. Patent Dec. 9, 2014 Sheet 8 Of 59 US 8,906,636 B2
8.3 %|
U.S. Patent Dec. 9, 2014 Sheet 12 Of 59 US 8,906,636 B2
.T?I,eTV INo.
§§ §§
eTV
Z?
U.S. Patent Dec. 9, 2014 Sheet 14 Of 59 US 8,906,636 B2
{{LGIRLÍTÐIH
SÃOneTeSÁT5)oT?
LO TZT Tº I9I L'8L TOZ TZZ U.S. Patent Dec. 9, 2014 Sheet 15 Of 59 US 8,906,636 B2
COLGIRI[10][H
SÃOeTII99TV!TV,OIausvg
IZ TL8 TOL IzI IL9I T& TOZ ILZ
U.S. Patent Dec. 9, 2014 Sheet 17 Of 59 US 8,906,636 B2
OHLOHRITTOIH
[9 [8 TOI U.S. Patent Dec. 9, 2014 Sheet 18 Of 59 US 8,906,636 B2
l J
{xx x:
x. s:
s
-: s s
s
yx s U.S. Patent Dec. 9, 2014 Sheet 19 Of 59 US 8,906,636 B2
W6GHRIQOIDH
U.S. Patent Dec. 9, 2014 Sheet 21 Of 59 US 8,906,636 B2
06OHRIÍT?INH
C)
AT5
|
U.S. Patent Dec. 9, 2014 Sheet 23 Of 59 US 8,906,636 B2
TeA () næT H
TÈA sTH neT I§§ SÄTT UTE)
TeA UTE) ©IA neT TÄT UIT5 eT. TeA ?I §ÅTT §ÅTT eT. eT nT9
OT 02 ZZ 9Z 8Z
U.S. Patent Dec. 9, 2014 Sheet 32 Of 59 US 8,906,636 B2
×
fg s COL
TÊ
U.S. Patent Dec. 9, 2014 Sheet 33 Of 59 US 8,906,636 B2
XIIIGIRLÍTÐIH
U.S. Patent Dec. 9, 2014 Sheet 36 of 59 US 8,906,636 B2
OS InN 719 849 2A9 19 O/9 699 899 A99 999 999 799 999 99 99 O99 699 899 199 999 999 799 999 299 99 Jeppe
U.S. Patent Dec. 9, 2014 Sheet 39 Of 59 US 8,906,636 B2
s & : U.S. Patent Dec. 9, 2014 Sheet 40 of 59 US 8,906,636 B2
s U.S. Patent Dec. 9, 2014 Sheet 41 of 59 US 8,906,636 B2
s
o o "si; S. co N. D. CO e S N N D 90 g N CO ear A. 3- sS CO Y s P SirS N ey st O N s N S o
r N S- co 9. s U.S. Patent Dec. 9, 2014 Sheet 42 of 59 US 8,906,636 B2
5:1~
9IGIRI[10IH
U.S. Patent Dec. 9, 2014 Sheet 43 of 59 US 8,906,636 B2
VLIGIRLÍTÐIH
IS
y
U.S. Patent Dec. 9, 2014 Sheet 50 of 59 US 8,906,636 B2
V6IGIRI[15)IH
peu?T-Tepun U.S. Patent Dec. 9, 2014 Sheet 51 of 59 US 8,906,636 B2
U.S. Patent Dec. 9, 2014 Sheet 53 of 59 US 8,906,636 B2
(I6IGIRLÍTÐIH
3. cy
U.S. Patent Dec. 9, 2014 Sheet 54 Of 59 US 8,906,636 B2
as
U.S. Patent Dec. 9, 2014 Sheet SS Of 59 US 8,906,636 B2
9|
IZGIRLÍTÐIH 9 U.S. Patent Dec. 9, 2014 Sheet 56 of 59 US 8,906,636 B2
ZZCHRI[15)IH
U.S. Patent Dec. 9, 2014 Sheet 58 Of 59 US 8,906,636 B2
16L-INNO?Iqn?osTI5 U.S. Patent Dec. 9, 2014 Sheet 59 of 59 US 8,906,636 B2
SZGIRI(101||H. US 8,906,636 B2 1. 2 HIGH LEVEL, EXPRESSION OF TER(R) (Wyeth), a Haemophilus influenzae type b vaccine. In RECOMBINANT TOXIN PROTEINS addition, CRM197 has potential use as a boosting antigen for C. diphtheria Vaccination and is being investigated as a car CLAIM OF PRIORITY rier protein for use in other vaccines. A method for high-level expression of CRM197 for This application is a divisional of U.S. patent application approved therapeutics and investigational use has not been Ser. No. 13/073,955, filed on Mar. 28, 2011 and issued as U.S. reported. CRM197 has been expressed in, e.g., C. diphthe Pat. No. 8,530,171 on Sep. 10, 2013, which claims priority riae, B. subtilis, and E. coli, at levels that range in the tens of under 35 U.S.C. S 119(e) to U.S. Provisional Application Ser. mg/L. A single dose of the Prevnar conjugate vaccine con No. 61/325,235 filed on Apr. 16, 2010, PCT/US 10/30573 10 tains about 20 ug of CRM197. Therefore, a method for eco filed on Apr. 9, 2010, and U.S. Provisional Application Ser. nomically producing CRM197 at levels of about 1 g/L or No. 61/319,152 filed on Mar. 30, 2010, and is a continuation more would greatly facilitate vaccine research and manufac in-part of PCT/US 10/30573, filed on Apr. 9, 2010, which claims priority to U.S. Provisional Application Ser. No. ture. 61/319,152 filed on Mar. 30, 2010. The contents of these 15 Cholera Toxin (CTX), produced by Vibrio cholera, a bac applications are hereby incorporated by reference in their terial pathogen that causes an infection characterized by diar entirety. rhea and vomiting, is also an ADP-ribosylating toxin. CTX is an oligomeric complex made up of six protein Subunits: a SEQUENCE LISTING single copy of the Cholera toxin A subunit (CTA), and five copies of the Cholera Toxin B subunit (CTB). The five B The instant application contains a Sequence Listing which Subunits, each weighing 12 kDa, form a five-membered ring. has been submitted in ASCII format via EFS-Web and is The A subunit has an A1 portion, CTA1, a globular enzyme hereby incorporated by reference in its entirety. Said ASCII that ADP-ribosylates G proteins, and an A2 chain, CTA2, that copy, created on Mar. 16, 2011, is named 38.1942.01.txt and is forms an extended alpha helix which sits Snugly in the central 156,975 bytes in size. 25 pore of the B subunit ring. This ring binds to GM1 ganglioside receptors on the host cell Surface, resulting in internalization BACKGROUND OF THE INVENTION of the entire complex. Once internalized, the CTA 1 chain is released by reduction of a disulfide bridge. CTA 1 is then Microbial toxin proteins are used in medicine, as immuno activated and catalyzes ADP ribosylation of adenylate gens for vaccination against the toxin-producing microbe and 30 cyclase. The resulting increase in adenylate cyclase activity as carrier proteins and adjuvants for other vaccines, and in increases cyclic AMP synthesis, which causes massive fluid scientific research as tools for studying molecular pathways. and electrolyte efflux and results in diarrhea. Diphtheria toxin (DT) is a proteinaceous toxin that is syn The B subunit of CTX, though relatively harmless, retains thesized and secreted by toxigenic strains of Corynebacte its ability to bind to the GM1 ganglioside receptor. CTB rium diphtheriae. Toxigenic strains contain a bacteriophage 35 therefore finds use in facilitating mucosal uptake of chemi lysogen carrying the toxin gene. DT is synthesized as a 535 cally or genetically conjugated foreign antigens. It has been amino-acid polypeptide, which undergoes proteolysis to demonstrated to induce both mucosal and systemic immu form the mature toxin. The mature toxin comprises two Sub nity, and is a candidate for use in edible vaccine production. units, A and B, joined by a disulfide bridge. The B subunit, Because of its binding preference, CTB also finds use as a formed from the C-terminal portion of intact DT enables 40 neuronal tracer. binding and entry of DT through the cell membrane and into Pertussis toxin (PTX) is an exotoxin and virulence factor the cytoplasm. Upon cell entry, the enzymatic A Subunit, produced by Bordetella pertussis, a bacterial pathogen of the formed from the N terminal portion of intact DT, catalyzes human respiratory tract that causes the disease whooping ADP ribosylation of Elongation Factor 2 (EF-2). As a result, cough. The pertussis holotoxin is a multi-subunit complex EF-2 is inactivated, protein synthesis stops, and the cell dies. 45 with an AB 5 structure. The enzymatically active A subunit Diphtheria toxin is highly cytotoxic; a single molecule can be (S1) is an ADP-ribosyltransferase that modifies the alpha lethal to a cell, and a dose of 10 ng/kg can kill animals and Subunit of several heterotrimeric G proteins in mammalian humans. cells, and the Boligomer (S2, S3, two copies of S4, and S5) The CRM197 protein is a nontoxic, immunologically binds glycoconjugate receptors on cells. The five subunits of cross-reacting form of DT. It has been studied for its potential 50 the toxin are expressed from the Pertussis Toxoid operon. use as a DT booster or vaccine antigen. CRM197 is produced Nontoxic variants of Pertussis toxin have been explored for by C. diphtheriae that has been infected by the nontoxigenic use in protective vaccines and as a vaccine adjuvant. There is phage B197toX-created by nitrosoguanidine mutagenesis of also a need for Pertussis toxin protein to use in research, e.g., the toxigenic corynephage f3. The CRM197 protein has the for studies of G protein signaling pathways. same molecular weight as DT but differs by a single base 55 Tetanus Toxin, produced by Clostridium tetani, is a neuro change (guanine to adenine) in the A Subunit. This single base toxin having a molecular weight of 150 kDa. It is made up of change results in an amino acid substitution (glutamic acid two parts: a 100 kDa heavy or B-chain and a 50 kDa light or for glycine) and eliminates the toxic properties of DT. A-chain. The chains are connected by a disulfide bond. The Conjugated polysaccharide vaccines that use CRM197 as a B-chain binds to disialogangliosides (GD2 and GD1b) on the carrier protein have been approved for human use. Vaccines 60 neuronal membrane. The A-chain, a Zinc endopeptidase, include: Menveo(R) (Novartis Vaccines and Diagnostics), a attacks the vesicle-associated membrane protein (VAMP). vaccine indicated for preventing invasive meningococcal dis The action of the A-chain stops the affected neurons from ease caused by Neisseria meningitidis Subgroups A, C.Y. and releasing the inhibitory neurotransmitters GABA (gamma W-135: Menjugate (Novartis Vaccines), a meningococcal aminobutyric acid) and glycine by degrading the protein Syn group C conjugate vaccine; and Prevnar R. (Wyeth Pharma 65 aptobrevin. The consequence of this is dangerous overactivity ceuticals, Inc.), a childhood pneumonia vaccine that targets in the muscles from the smallest stimulus—the failure of seven serotypes of Streptococcus pneumoniae, and HibTI inhibition of motor reflexes by sensory stimulation. This US 8,906,636 B2 3 4 causes generalized contractions of the agonistandantagonist In other embodiments, the recombinant toxin protein is musculature, termed a tetanic spasm. CRM197, Diphtheria Toxin, Cholera holotoxin, Cholera Tetanus Toxin Fragment C (Tet C or TTC) is a 50 kD Toxin B. Pertussis toxin, Tetanus Toxin Fragment C, or C. polypeptide generated by protease cleavage (e.g., with difficile Toxin B. papain) of Tetanus toxin, or through recombinant expression In certain embodiments, the recombinant protein is pro of the fragment. It corresponds to the 451 amino acids at the duced at a yield of soluble and/or active toxin protein of about C-terminus (amino acid positions 865-1315). 0.2 grams per liter to about 12 grams per liter. In specific Fragment C has been shown to be non-toxic. Because it embodiments, the yield of soluble and/or active toxin protein binds to neurons with high specificity and affinity, TTC finds is about 0.2 g/L, about 0.3 g/L, about 0.4 g/L, about 0.5g/L, 10 about 0.6 g/L, about 0.7 g/L, about 0.8 g/L, about 0.9 g/L, use as a targeting molecule for neuronal drug delivery or for about 1 g/L, about 1.5 g/L, about 2 g/L, about 2.5g/L, about research purposes. TTC protein is also potentially useful as a 3 g/L, about 3.5g/L, about 4 g/L, about 4.5g/L, about 5g/L, vaccine carrier protein and for use in a vaccine to protect about 5.5 g/L, about 6 g/L, about 6.5 g/L, about 7 g/L, about against C. tetani infection. 7.5g/L, about 8 g/L, about 8.5g/L, about 9 g/L, about 9.5g/L, Clostridium difficile Toxin B (TcdB) is a virulence factor 15 about 10 g/L, about 10.5 g/L, about 11 g/L, about 12 g/L, produced by Clostridium difficile, which causes hospital about 0.2 g/L to about 0.5g/L, about 0.2 g/L to about 1 g/L, acquired diarrhea and pseudomembranous colitis. TcdB, and about 0.2 to about 2 g/L, about 0.3 g/L to about 0.6 g/L, about a second large clostridial toxin, TcdA, are involved in the 0.3 g/L to about 1 g/L, about 0.3 to about 2 g/L, about 0.4 to development of pseudomembranous colitis. about 0.7 g/L, about 0.4 to about 1 g/L about 0.4 to about 2 TcdB is a glucosylating toxin of about 270 kD, and can be g/L, about 0.4 to about 3 g/L, about 0.5 g/L to about 1 g/L, divided into enzymatic, translocation and receptor binding about 0.5 g/L to about 2 g/L, about 0.5 g/L to about 3 g/L, domains. The first 546 amino acids of TcdB contain the about 0.5 g/L to about 4 g/L, about 0.5 g/L to about 5 g/L, enzymatic region, which is followed by a putative transloca about 0.5 g/L to about 6 g/L, about 0.5 g/L to about 7 g/L, tion and receptor-binding domain. TcdB has potential use as about 0.5 g/L to about 8 g/L, about 0.5 g/L to about 9 g/L, a protective vaccine for C. difficile infection, as well as in 25 about 0.5 g/L to about 10 g/L, about 0.5 g/L to about 11 g/L, diagnostic tests and their development. about 0.5 g/L to about 12 g/L, about 1 g/L to about 2 g/L. Exotoxin A (ETA or PE) of Pseudomonas aeruginosa is a about 1 g/L to about 3 g/L, about 1 g/L to about 4 g/L, about Type II ADPRT. Like its family members Diphtheria toxin 1 g/L to about 5 g/L, about 1 g/L to about 6 g/L, about 1 g/L and Cholera Toxin, it inhibits protein synthesis by the ADP to about 7 g/L, about 1 g/L to about 8 g/L, about 1 g/L to about ribosylation of cellular elongation factor 2. P. aeruginosa 30 9 g/L, about 1 g/L to about 10 g/L, about 1 g/L to about 11 g/L. about 1 g/L to about 12 g/L, about 2 g/L to about 3 g/L, about Exotoxin A exists as a monomer, consisting of a single 2 g/L to about 4 g/L, about 2 g/L to about 5g/L, about 2 g/L polypeptide chain of 613 amino acids (66. Kd). to about 6 g/L, about 2 g/L to about 7 g/L, about 2 g/L to about ETA is potentially useful as a vaccine conjugate. Nontoxic 8 g/L, about 2 g/L to about 9 g/L, about 2 g/L to about 10 g/L. mutants of ETA have been studied as vaccine conjugates for 35 about 2 g/L to about 11 g/L, about 2 g/L to about 12 g/L, about vaccinations that protect against Staphylococcus aureus, 3 g/L to about 4 g/L, about 3 g/L to about 5g/L, about 3 g/L malaria, and Salmonella Tiphi. to about 6 g/L, about 3 g/L to about 7 g/L, about 3 g/L to about Producing these toxins in amounts Sufficient to meet 8 g/L, about 3 g/L to about 9 g/L, about 3 g/L to about 10 g/L, expanding needs has presented significant challenges. When about 3 g/L to about 11 g/L, about 3 g/L to about 12 g/L, about made in conventional protein overexpression systems, the 40 4 g/L to about 5 g/L, about 4 g/L to about 6 g/L, about 4 g/L toxin proteins are recovered in active form only at very low to about 7 g/L, about 4 g/L to about 8 g/L, about 4 g/L to about concentration due to degradation, improper folding, or both, 9 g/L, about 4 g/L to about 10 g/L, about 4 g/L to about 11 g/L. depending on the specific characteristics, e.g., size and sec about 4 g/L to about 12 g/L, about 5 g/L to about 6 g/L, about ondary structure, of the toxin. Therefore, methods for pro 5 g/L to about 7 g/L, about 5 g/L to about 8 g/L, about 5 g/L ducing large amounts of these toxins, in soluble and/or active 45 to about 9 g/L, about 5 g/L to about 10 g/L, about 5 g/L to form, and at low cost is needed. about 11 g/L, about 5 g/L to about 12 g/L, about 6 g/L to about 7 g/L, about 6 g/L to about 8 g/L, about 6 g/L to about 9 g/L, SUMMARY OF THE INVENTION about 6 g/L to about 10 g/L, about 6 g/L to about 11 g/L, about 6 g/L to about 12 g/L, about 7 g/L to about 8 g/L, about 7 g/L The present invention relates to a method for producing a 50 to about 9 g/L, about 7 g/L to about 10 g/L, about 7 g/L to recombinant toxin protein in a Pseudomonad host cell, said about 11 g/L, about 7 g/L to about 12 g/L, about 8 g/L to about method comprising: ligating into an expression vector a 9 g/L, about 8 g/L to about 10 g/L, about 8 g/L to about 11 g/L, nucleotide sequence encoding a toxin protein; transforming about 8 g/L to about 12 g/L, about 9 g/L to about 10 g/L, about the Pseudomonas host cell with the expression vector; and 9 g/L to about 11 g/L, about 9 g/L to about 12 g/L, about 10 55 g/L to about 11 g/L, about 10 g/L to about 12 g/L, or about 11 culturing the transformed Pseudomonas host cell in a culture g/L to about 12 g/L. media suitable for the expression of the recombinant toxin In embodiments, the nucleotide sequence encoding the protein; wherein the recombinant toxin protein is CRM197, toxin protein is fused to a secretion signal coding sequence Diphtheria Toxin, Cholera holotoxin, Cholera Toxin B. Per that when expressed directs transfer of the toxin protein to the tussis toxin, Tetanus Toxin Fragment C. C. difficile Toxin B, 60 periplasm. In embodiments, the host cell is defective in the or P aeruginosa Exotoxin A. expression of at least one protease or the host cell overex In embodiments, the recombinant toxin protein is Cholera presses at least one folding modulator, or a combination Toxin B, Cholera holotoxin, Pertussis toxin, Tetanus Toxin thereof. Fragment C. C. difficile Toxin B, or Paeruginosa Exotoxin A. In embodiments, the recombinant toxin protein is In other embodiments, the recombinant toxin protein is 65 CRM197 and the host cell is defective in the expression of Cholera Toxin B, Cholera holotoxin, Pertussis toxin, Tetanus HslU, HslV. Prc1, DegP1, DegP2, and AprA. In related Toxin Fragment C, or C. difficile Toxin B. embodiments, the recombinant toxin protein is fused to a US 8,906,636 B2 5 6 secretion leader that is AZu, IbpS31A, Cup A2, PbpA20V, or units (AU), the pH of the culture is from about 6 to about 7.5, Pbp. In embodiments, the recombinant toxin protein is and the growth temperature is about 20 to about 35°C. CRM197 and the host cell is defective in the expression of In embodiments, the host cell is a Pseudomonas cell. In HslU and HslV, or Prcl, or DegP1, or DegP2, or AprA. In related embodiments, the host cell is Pseudomonas fluore specific embodiments, the recombinant toxin protein is 5 SCéS. CRM197 and the host cell is defective in the expression of In embodiments of the invention, the nucleotide sequence Serralysin, HslU, HslV. Prc1, DegP1, DegP2, or AprA, or the has been optimized for expression in the Pseudomonad host host cell overexpresses DsbA, DsbB, DsbC, and DsbD. In cell. In related embodiments, the nucleotide sequence has embodiments, the host cell overexpresses DsbA, DsbB, been optimized for expression in the Pseudomonas host cell. DsbC, and DsbD, and the recombinant toxin protein is fused 10 In other related embodiments, the nucleotide sequence has to the secretion leader AZu. In embodiments, the host cell is been optimized for expression in the Pseudomonas fluore defective in the expression of Serralysin, and the recombinant scens host cell. toxin protein is fused to the secretion leader Pbp or AZu. In In embodiments, the Pertussis toxin is wild-type or S1 embodiments, the host cell is defective in the expression of E129A R9K. In embodiments, the P. aeruginosa Exotoxin A HslU and HslV, and the recombinant toxin protein is fused to 15 is wild-type, CRM66, or rEPA. the secretion leader Pbp or AZu. In embodiments, the recom In embodiments of the invention, the expression vector binant toxin protein is CRM197, the host cell is wild-type and further comprises a tag sequence adjacent to the coding wherein the recombinant toxin protein is fused to the secre sequence for the Secretion signal. In embodiments, the tion leader Pbp or AZu. In embodiments, the recombinant expression vector further comprises a tag sequence adjacent toxin protein is CRM197 and the recombinant toxin protein is to the coding sequence for the toxin protein. fused to the secretion leader AZu, Pbp, IbpS31A, Cup A2, or The present invention also provides a recombinant toxin PbpA20V. protein produced according to the methods described herein. In other embodiments, the recombinant toxin protein is In embodiments, the recombinant toxin protein is CRM197, Cholera Toxin B and the host cell is defective in the expres Diphtheria Toxin, Cholera holotoxin, Cholera Toxin B. Per sion of Lon, La, and AprA, or the host cell is defective in the 25 tussis Toxin, Tetanus Toxin fragment C. C. difficile Toxin B, expression of HslU, HslV. Prc1, DegP1, DegP2, and AprA. In or P. aeruginosa Exotoxin A. In embodiments, the Exotoxin related embodiments, the host cell is defective in the expres A is wild-type, CRM66, or rEPA. In certain embodiments, the sion of Lon, La, and AprA and wherein the recombinant toxin recombinant toxin protein is produced in a strain of Pfluo protein is fused to the secretion leader Pbp A20V. rescens identified herein as producing a highyield of the toxin In other embodiments, the recombinant toxin protein is 30 or producing high quality toxin. In certain embodiments, the Pertussis toxin S1 E129A R9K and the host cell is defective in recombinant toxin protein is produced in a strain of Pfluo the expression of Lon, La, and AprA; GrpE. DnaK, and rescens described hereinas producing the highest yield of the DnaJ: HtpX; RXFO1590; or ppiB (RXF05345). In related toxin protein. In other embodiments, the recombinant toxin embodiments, the recombinant toxin protein is fused to its protein is produced in a strain described hereinas one used for native secretion leader. 35 fermentation production of the toxin. In other embodiments, the recombinant toxin protein is Tetanus Toxin C and the host cell is defective in the expres INCORPORATION BY REFERENCE sion of HslU, HslV. Prc1, DegP1, DegP2, and AprA. In related embodiments, the recombinant toxin protein is fused All publications, patents, and patent applications men to the secretion leader DsbC, Pbp A20V, or Cup A2. 40 tioned in this specification are herein incorporated by refer In other embodiments, the recombinant toxin protein is ence to the same extent as if each individual publication, Tetanus Toxin C and the host cell is defective in the expres patent, or patent application was specifically and individually sion of Lon, La, and AprA. In related embodiments, the indicated to be incorporated by reference. recombinant toxin protein is fused to the secretion leader DsbA. 45 BRIEF DESCRIPTION OF THE DRAWINGS In other embodiments, the recombinant toxin protein is Tetanus Toxin C and the host cell is defective in the expres The novel features of the invention are set forth with par sion of GrpE. DnaK, and DnaJ. In related embodiments, the ticularity in the appended claims. A better understanding of recombinant toxin protein is fused to the secretion leader the features and advantages of the present invention will be NikA. 50 obtained by reference to the following detailed description In other embodiments, the recombinant toxin protein is C. that sets forth illustrative embodiments, in which the prin difficile Toxin Band the host cell is defective in the expression ciples of the invention are utilized, and the accompanying of: HtpX: DegP1; HslU, HslV. Prc1 and Prc2; or Lon and drawings. DegP2, or the host cell is both defective in the expression of FIG. 1A to 1C. CRM197 Amino Acid and DNA Lon, Prc1, DegP2, AprA and overexpresses DegP2S219A. 55 Sequences. 1A. Amino acid sequence (SEQID NO: 1). 1B. In embodiments, the activity of the recombinant toxin pro First segment of an optimized DNA sequence (SEQID NO:2) tein is measured in an activity assay, wherein about 40% to encoding the CRM197 protein, with translation shown. 1C. about 100% of the soluble toxin protein produced is deter Second segment of an optimized DNA sequence (SEQ ID mined to be active. In related embodiments, the activity assay NO:2) encoding the CRM197 protein, with translation is an immunological assay, a receptor-binding assay, or an 60 shown. This optimized sequence is a non-limiting example of enzyme assay. an optimized sequence useful in the methods of the present In embodiments of the invention, the expression vector invention. comprises a lac derivative promoter operatively linked to the FIG. 2. High Throughput Expression Analysis of protein coding sequence, and wherein the culturing com CRM197. CRM197 protein expressed using the DNA prises induction of the promoter using IPTG at a concentra 65 sequence shown in FIG. 1B was analyzed using capillary gel tion of about 0.02 to about 1.0 mM, the cell density at induc electrophoresis (SDS-CGE). Soluble fractions of 40 tion is an optical density of about 40 to about 200 absorbance CRM197-expression strains tested are shown in a gel-like US 8,906,636 B2 7 8 image generated from the SDS-CGE data. Strain names as shown in a gel-like image generated from the SDS-CGE data. described in Table 10 are listed above each lane. P. fluore Strain names as described in Table 15 are listed above each scens-expressed CRM197 migrated as a single band at ~58 lane. Induced Tetanus Toxin C Fragment migrated as a single kDa on SDS-CGE (arrow at left). Molecular weight markers band at -51.6 kDa on SDS-CGE (arrow at left). Molecular in first and last lanes are 16, 20, 29, 48, 69 and 119 kDa. 5 weight markers in first and last lanes are 16, 20, 29.48, 69 and FIG.3.Cholera Toxin BAmino Acid and DNA Sequences. 119 kDa. A. Amino acid sequence (SEQID NO: 22). B. An optimized FIG. 11A to 11M.TcdBAmino Acid and DNA Sequences. DNA sequence (SEQ ID NO. 23) encoding the CTB protein, 11A. First segment of amino acid sequence (SEQID NO:32). with translation. This optimized sequence is a non-limiting 11B. Second segment of amino acid sequence (SEQ ID example of an optimized sequence useful in the methods of 10 NO:32). 11C. Third segment of amino acid sequence (SEQ the present invention. ID NO:32). 11D. Fourth segment of amino acid sequence FIG. 4. High Throughput Expression Analysis of Cholera (SEQID NO:32). 11E. Fifth segment of amino acid sequence Toxin B. Cholera Toxin B protein expressed using the DNA (SEQID NO:32). 11F. Sixth segment of amino acid sequence sequence shown in FIG. 3B was analyzed using capillary gel (SEQ ID NO:32). 11G. First segment of an optimized DN electrophoresis (SDS-CGE). Soluble fractions from 40 chol 15 sequence encoding the TcdB protein, with translation (SE era toxin-expression strains tested are shown in a gel-like ID NO:33). 11H. Second segment of an optimized DN image generated from the SDS-CGE data. Strain names as sequence encoding the TcdB protein, with translation (SE described in Table 11 are listed above each lane. Induced CTB ID NO:33). 11I. Third segment of an optimized DN migrated as a single band at ~11.5 kDa on SDS-CGE (arrow sequence encoding the TcdB protein, with translation (SE at left). Molecular weight markers in first and last lanes are ID NO:33). 11J. Fourth segment of an optimized DN 16, 20, 29, 48, 69 and 119 kDa. sequence encoding the TcdB protein, with translation (SE FIG. 5. Pertussis Toxoid Operon. BPETOX S1-R9K & ID NO:33). 11K. Fifth segment of an optimized DN E129A, having 4210 basepairs, is shown. sequence encoding the TcdB protein, with translation (SE FIG. 6A to 6E. DNA Sequence of the Pertussis Toxoid. 6A. ID NO:33). 11L. Sixth segment of an optimized DN First segment of the Pertussis toxin S1 R9K E129A DNA 25 sequence encoding the TcdB protein, with translation (SE sequence with translation is shown (SEQ ID NO:24). The ID NO:33). 11M. Seventh segment of an optimized DN sequence is derived from Genebank entry M13223. Subunits sequence encoding the TcdB protein, with translation (SEQ S1-S5 and signal sequences are indicated above the ID NO:33). This optimized sequence is a non-limiting sequences. The R9K and E129A mutations in S1 are under example of an optimized sequence useful in the methods of lined. Encoded proteins are disclosed as SEQID NOS 25, 26, 30 the present invention. 28, 29, and 27, respectively, in order of appearance. 6B. FIG. 12. TcdB Expression. TcdB expressed in Pfluore Second segment of the Pertussis toxin S1 R9K E129A DNA scens was analyzed using capillary gel electrophoresis (SDS sequence with translation is shown (SEQ ID NO:24). 6C. CGE). Soluble fractions from 24 TcdB-expression strains Third segment of the Pertussis toxin S1 R9K E129A DNA tested are shown in a gel-like image generated from the SDS sequence with translation is shown (SEQ ID NO:24). 6D. 35 CGE data. Strain names as described in Table 18 as well as Fourth segment of the Pertussis toxin S1 R9K E129A DNA null extract and reference standard (List Biologicals) are sequence with translation is shown (SEQ ID NO:24). 6E. listed above each lane. Induced TcdB migrated as a single Fifth segment of the Pertussis toxin S1 R9K E129A DNA band at -300 kDa on SDS-CGE (arrow at left). Molecular sequence with translation is shown (SEQID NO:24). weight markers in first and last lanes are 16, 20, 29.48, 69 and FIG. 7A to 7E. Amino Acid Sequences of Pertussis Toxoid 40 119 kDa. Subunits. Secretion signals are underlined. 7A. S1 subunit FIG. 13A to 13B. Exotoxin A Amino Acid Sequence. 13A. (R9K E129A) (SEQ ID NO:25). 7B. S2 subunit (SEQ ID First segment of the amino acid sequence of P aeruginosa NO:26). 7C. S3 subunit (SEQ ID NO:27). 7D. S4 subunit Exotoxin A is shown (SEQID NO:34). 13B. Second segment (SEQ ID NO:28).7E. S5 subunit (SEQID NO:29). of the amino acid sequence of P aeruginosa Exotoxin A is FIG.8. Western blot analysis of Pertussis Toxoid expres 45 shown (SEQID NO:34). Three Exotoxin A proteins are indi sion samples. Strain names are listed above each lane. cated by the drawing: wild-type, CRM66, and rEPA. In vari Induced PtX migrated as multiple bands range from 11 to 26 ant CRM66, His 426 (bold, underlined text) is replaced by a kDa (S1: 26.1 Kda, S2:20.9 Kda, S3: 21.8 KDa, S4 (2x): 12 Tyras indicated above the sequence. In rEPA, Glu553 (bold, KDa, S5: 11 KDa) A. Reduced samples. B. Non-reduced underlined text) is deleted as indicated above the sequence. samples. Both panels: Lane 1—molecular weight markers 50 FIG. 14. Soluble Cholera Toxin B Production in Pfluore (10, 15, 20, 25.37, 50, 75, 100, 150,250kDa); Lane 2 Null: scens Fermentation Cultures. SDS-CGE Analysis. Lane 1-16, Lane 3—strain 321; Lane 4-strain 322; Lane 5—strain 323; 20, 29, 48, 69 and 119 kDa molecular weight markers. Lanes Lane 6—strain 324; Lane 7 strain 325; Lane 8—strain 326; 2 and 4-pre-induction samples and lanes 3 and 5 post-induc Lane 9—strain 327; Lane 10 strain 328. tion samples, respectively, of PS538-088 U5 and U6 fermen FIG. 9A to 9C. Tetanus Toxin C Amino Acid and DNA 55 tations expressing Cholera Toxin B, indicated by arrow at Sequences. 9A. Amino acid sequence (SEQID NO:30).9B. right. First segment of an optimized DNA sequence encoding the FIG. 15. Soluble Tetanus Toxin Fragment C Production in Tetanus Toxin C protein, with translation (SEQID NO:31). Pfluorescens Fermentation Cultures. A. SDS-CGE Analysis. 9.C. Second segment of an optimized DNA sequence encod Lane 1-16, 20, 29, 48, 69 and 119 kDa molecular markers. ing the Tetanus Toxin C protein, with translation (SEQ ID 60 Lanes 2, 3 and 4 are post-induction samples of PS538-529 U1 NO:31). This optimized sequence is a non-limiting example PS538-546 U5 and PS538-547 U7 fermentations, respec of an optimized sequence useful in the methods of the present tively, expressing Tetanus Toxin Fragment C, indicated by invention. arrow at right. B. Western Blot Analysis. Fermentation FIG. 10. Tetanus Toxin C Fragment Expression. Tetanus samples from strains PS538-538 (U1 and U2), PS538-548 Toxin C Fragment expressed in Pfluorescens was analyzed 65 (U3 and U4), PS538-558 (U5 and U6) and PS538-568 (UT using capillary gel electrophoresis (SDS-CGE). Soluble frac and U8) were evaluated by Western blot. Fermentation unit tions from 40 tetanus toxin-expression strains tested are and hours post induction (IO, I8, I24) are indicated above each US 8,906, 636 B2 9 10 lane. Molecular weight (MW) standards are shown on the left FIG. 22. Western Blot of Soluble rEPA Production in P of the blot and Tetanus Toxin C reference standard (Std; List fluorescens Fermentation Cultures. Soluble rEPA expressed Biological, Catil 193) is shown on the right. Blots were probed infermentation cultures of Pfluorescens were analyzed using with Polyclonal Anti-Tetanus Toxin C Fragment, derived in Western blot analysis. Soluble fractions from fermentations Rabbit (Abcam, Cath: ab34890) followed by Anti-Rabbit IgG 5 of expression strains PS538-1633 (u1), PS538-1640 (u3 and Peroxidase, derived in Goat (Pierce, Cath: 3.1460). Immu u5) and PS538-1670 (u6 and u8) at 0 and 24 hours post nopure Metal Enhanced DAB (Pierce 34065) was used for induction are shown in a Western blot analysis using an anti detection. body specific for P aeruginosa Exotoxin A. Mw-molecular FIG. 16. Soluble C. difficile B Toxin Protein Production in weight standards. Std-rEPA standard. Pfluorescens Fermentation Cultures. Lane 1-16, 20, 29, 48, 10 FIG. 23. SDS-CGE Gel-like Image of Soluble CRM197 69 and 119 kDa molecular weight markers. The marker sizes Production in Pfluorescens Fermentation Cultures. CRM197 are also indicated in their respective positions at the right, expressed in fermentation cultures of Pfluorescens was ana based on migration in Lane 1. Lanes 2, 3 and 4 are post lyzed using capillary gel electrophoresis (SDS-CGE). induction samples of PS538-671 U5 and U6, and PS538-674 Soluble fractions from various fermentations of expression U7 fermentations, respectively, expressing C. difficile B 15 strains PS538-772 (u1 and u2), PS538-776 (u3 and u5) and Toxin Protein, indicated by arrow at right. PS538-782 (u6 and u7) at various times post-induction (0, 16, FIG. 17A to 17 E. DNA Sequence of Wild-Type Pertussis 21 and 23 hours) tested are shown in a gel-like image gener Toxoid. 17A. First segment of the wild-type Pertussis toxin ated from the SDS-CGE data. Mw-molecular weight stan DNA sequence with translation is shown (SEQ ID NO:35). dards (16, 20, 29, 48, 68, and 119 kilodaltons). 17B. Second segment of the wild-type Pertussis toxin DNA 20 FIG. 24. Soluble CRM197 Production Trends in Pfluore sequence with translation is shown (SEQ ID NO:35). 17C. scens Fermentation Cultures. Soluble CRM197 expression Third segment of the wild-type Pertussis toxin DNA levels as determined by SDS-CGE from the different strains sequence with translation is shown (SEQ ID NO:35). 17D. (PS538-772, PS538-776 and PS538-782) in their respective Fourth segment of the wild-type Pertussis toxin DNA fermentations (ul, u2, u3, u6 and u7) are plotted against sequence with translation is shown (SEQ ID NO:35). 17E. 25 post-induction times. Fifth segment of the wild-type Pertussis toxin DNA sequence FIG. 25. Western Blot of Soluble CRM197 Production in P with translation is shown (SEQID NO:35). The sequence is fluorescens Fermentation Cultures. CRM197 expressed in from Genebank entry M13223. Subunits S1-S5 and signal fermentation cultures of Pfluorescens were analyzed using sequences are indicated above the sequences. The encoded Western blot analysis. Soluble fractions from various fermen proteins are disclosed as SEQID NOS 41-45, respectively, in 30 tations of expression strains PS538-772 (u1 and u2), PS538 order of appearance. 776 (u3 and us) and PS538-782 (u6 and u7) at various times FIG. 18A to 18B. Amino Acid and DNA Sequence of post-induction (0, 16, 21 and 23 hours) tested are shown in a Wild-Type Diphtheria toxin. 18A. Amino acid sequence Western blot analysis using a diphtheria toxin specific anti (SEQ ID NO:36). 18B. An optimized DNA sequence (SEQ body. Mw-molecular weight standards (37.50, 75, 100, 150, ID NO:37) encoding the DT protein, with translation shown. 35 and 250 kilodaltons). STD=CRM197standard. This optimized sequence is a non-limiting example of an optimized sequence useful in the methods of the present DETAILED DESCRIPTION OF THE INVENTION invention. The encoded protein is disclosed as residues 1-320 of SEQID NO:36. Toxins FIG. 19A to D. Amino Acid and DNA Sequence of Cholera 40 ADP-Ribosylating Toxins Holotoxin. 19A. CTA amino acid sequence (SEQID NO:38), ADP-ribosylating toxins (ADPRTs) facilitate scission of with secretion leader (underlined) (AE003852; Protein ID the N-glycosyl bond between nicotinamide and the N-ribose AAF946.14.1). 19B. CTBamino acid sequence (SEQID NO: of NAD and transfer the ADP-ribose moiety to target pro 39), with secretion leader (underlined) (GenBankAE003852; teins. ADPRTs are classified into four families based on their Protein ID AAF946.13.1). 19C. First segment of CTX DNA 45 respective targets. Type I ADPRTs target heteromeric GTP sequence (SEQID NO:40) indicating the A and B subunits, binding proteins. They include Cholera Toxin (CTX), Pertus with translation shown (Genbank AE003852). 19D. Second sis toxin (PTX), and Escherichia coli heat-labile enterotoxin segment of CTX DNA sequence (SEQID NO:40) indicating (LT). Type II ADPRTs (Diphtheria toxin and Pseudomonas the A and B subunits, with translation shown (Genbank Exotoxin A) modify elongation factor 2 (EF2). Type III AE003 852). The encoded proteins are disclosed as SEQ ID 50 ADPRTs (Clostridium botulinum C3 exoenzyme) ADP-ribo NOS 38 and 39, respectively, in order of appearance. sylate small GTP-binding proteins. Type IV ADPRTs ADP FIG. 20. SDS-CGE Gel-like Image of Soluble rEPA Pro ribosylate actin. These actin-specific ADPRTs include a fam duction in P. fluorescens Fermentation Cultures. Soluble ily of binary toxins comprising C. botulinum C2 toxin, C. rEPA expressed in fermentation cultures of Pfluorescens was perfingens L-toxin, C. difficile toxin (a toxin distinct from analyzed using capillary gel electrophoresis (SDS-CGE). 55 TcdA and TcdB, described by Popoff, et al., 1988, “Actin Soluble fractions from fermentations of expression strains specific ADP-ribosyltransferase produced by a Clostridium PS538-1633 (u1 and u2), PS538-1640 (u3 and us) and difficile strain.” Infection and Immunity 56(9): 2299-2306, PS538-1670 (u6, u7 and u8) at 0 and 24 hours post-induction incorporated herein by reference), C. Spiroforme toxin, and tested are shown in a gel-like image generated from the SDS Bacillus cereus vegetative insecticidal protein (VIP). CGE data. Mw-molecular weight standards (16, 20, 29, 48, 60 The structures of several enzymatic components from each and 69 kilodaltons). type of ADPRT have been determined with or without NAD, FIG. 21. Soluble rEPA Production Trends in Pfluorescens and are discussed by, e.g., Tsuge, et al., 2008, "Structural Fermentation Cultures. Soluble rEPA expression levels, as basis of actin recognition and arginine ADP-ribosylation by determined by SDS-CGE analysis of strains (PS538-1633, Clostridium perfingens-toxin.” PNAS 105(21):7399-7404, PS538-1640 and PS538-1670) in their respective fermenta- 65 incorporated herein by reference. Typical actin-specific tions (ul, u2, u3, u6, u7 and u8), are plotted against post ADPRTs possess two similar domains: the C domain, which induction times. is essential for catalytic activity; and the N domain, which is US 8,906,636 B2 11 12 important for the interaction with the binding and transloca The nucleotide sequence may be prepared using the tech tion subunit. By contrast, SpVB from Salmonella and the type niques of recombinant DNA technology (described by, e.g., III ADPRTC3 have only one ADP-ribosyltransferase domain Sambrook et al. Molecular Cloning, a Laboratory Manual, and lack the N-terminal adaptor domain. In all type IV Cold Spring Harbor Laboratory Press, 1989), and also by ADPRTs, the EXE motif, including two key glutamate resi 5 site-directed mutagenesis, based on the known DT nucleotide dues, is present at the catalytic center. The former glutamate sequence of the wildtype structural gene for Diphtheria toxin of the EXE motif is thought to be a key residue for ADP carried by corynebacteriophage B. (See, e.g., Greenfield, et ribosyltransferase, which is deprotonated from Arg-177 in al., 1993, "Nucleotide Sequence of the Structural Gene for actin. The latter glutamate forms a hydrogen bond with the Diphtheria toxin Carried by Corynebacteriophage 18. Proc O'2 on N-ribose, which is thought to stabilize the oxocarbe 10 Nat Acad Sci 80:6953-7, incorporated herein by reference.) nium cation. The nucleotide sequence can be optimized as described else ADPRTs are further described by Barth, et al., 2004, where herein. “Binary Bacterial Toxins: Biochemistry, Biology, and Appli In embodiments of the present invention, CRM197 or DT cation of Common Clostridium and Bacillus Proteins.” are produced using any of the host strains described herein in Microbiology and Molecular Biology Reviews 68(3):373 15 Example 1, in combination with any of the expression vectors 402; Mueller-Dieckmann, et al., “Structure of mouse ADP (plasmids) described in Example 1. In embodiments, the ribosylhydrolase 3 (mARH3).” Acta Cryst F64:156-162: nucleic acid sequence is optimized for expression in the Kulich, et al., 1995, “Expression of Recombinant Exoenzyme Pseudomonad host cell. In embodiments, the expression vec S of Pseudomonas aeruginosa. Infection and Immunity tors used contain constructs expressing any of the secretion 63(1):1-8; Sakurai, et al., 2009, "Clostridium perfingens leaders described in Table 8 and Table 3 fused to the recom Iota-Toxin: Structure and Function. Toxins 1:208-228; and binant CRM197 or DT protein. In embodiments, the native Schirmer, et al., 2002, “The ADP-ribosylating Mosquitocidal secretion leader is used. In certain embodiments, the Toxin from Bacillus sphaericus. The Journal of Biological CRM197 or DT protein is expressed with a tag, e.g., a puri Chemistry 277(14): 11941-11948, all incorporated herein by fication tag. In embodiments, the methods of the invention are reference. 25 used to produce CRM197 or DTata yield of about 0.5g/L to In embodiments of the present invention, a recombinant at least about 12 g/L. toxin protein selected from a group including ADPRTs is Cholera Toxin produced. In embodiments, the group of ADPRTs consists of Cholera toxin (CTX), produced by Vibrio cholera, is also CTX (CTA and/or CTB), PTX, DT (CRM197 and/or WT), an ADP-ribosylating toxin. The Cholera toxin (CTX) is an and Pseudomonas Exotoxin A. In embodiments, the group of 30 oligomeric complex made up of six protein Subunits: a single ADPRTs consists of CTX(CTA and/or CTB), PTX, and copy of the Cholera toxin A subunit (CTA), and five copies of Pseudomonas Exotoxin A. In other embodiments, a recom the Choleratoxin B subunit (CTB). The five B subunits, each binant toxin protein selected from a group including Type I weighing 12 kDa, form a five-membered ring. The A subunit ADPRTs is produced. In embodiments, the group of Type I has an A1 portion, CTA1, a globular enzyme that ADP-ribo ADPRTs consists of CTX (CTA and/or CTB), and PTX. In 35 Sylates G proteins, and an A2 chain, CTA2, that forms an other embodiments, a recombinant toxin protein selected extended alpha helix which sits Snugly in the central pore of from a group including Type II ADPRTs is produced. In the B subunit ring. This ring binds to GM1 ganglioside recep embodiments, the group of Type II ADPRTs consists of DT tors on the host cell Surface, resulting in internalization of the (CRM197 and/or WT), and Pseudomonas Exotoxin A. In entire complex. Once internalized, the CTA 1 chain is released other embodiments, a recombinant toxin protein selected 40 by reduction of a disulfide bridge. CTA 1 is then activated and from a group including Type IV ADPRTs is produced. In catalyzes ADP ribosylation of adenylate cyclase. The result embodiments, the Type IV ADPRT is TcdB. ing increase in adenylate cyclase activity increases cyclic CRM197 and DT AMP synthesis, which causes massive fluid and electrolyte Cross-reacting material 197 (CRM197) is a Diphtheria efflux and results in diarrhea. toxin (DT) variant produced from a DT gene having a mis 45 The B subunit of CTX, though relatively harmless, retains sense mutation. DT is an ADP-ribosylating toxin: CRM197 its ability to bind to the GM1 ganglioside receptor. CTB lacks the ADP-ribosyltransferase (ADPRT) activity of DT, therefore finds use in facilitating mucosal uptake of chemi and is thus nontoxic. The gene for CRM197 has a single base cally or genetically conjugated foreign antigens. It has been Substitution, resulting in the incorporation of glutamic acid demonstrated to induce both mucosal and systemic immu instead of glycine at residue 52. (See, e.g., Bishai, et al., 1987, 50 nity, and is a candidate for use in edible vaccine production. “High-Level Expression of a Proteolytically Sensitive Diph Because of its binding preference, CTB also finds use as a theria toxin Fragment in Escherichia coli.” J. Bact. 169(11): neuronal tracer. 5140-51, Giannini, et al., 1984, “The Amino-Acid Sequence The use of CTB, as well as its structural features, have been of Two Non-Toxic Mutants of Diphtheria toxin: CRM45 and described, e.g., by: Nozoye, et al., 2009, “Production of CRM197. Nucleic Acids Research 12(10): 4063-9, and Gen 55 Ascaris suum AS14 Protein and Its Fusion Protein with Chol Bank Acc. No. 1007216A, all incorporated herein by refer era Toxin B Subunit in Rice Seeds.” Parasitology 995-1000; ence.) Harakuni, et al., 2005. “Heteropentameric Cholera Toxin B CRM197 protein may be prepared at low levels by methods Subunit Chimeric Molecules Genetically Fused to a Vaccine known in the art or by expression in C. diphtheriae or other Antigen Induce Systemic and Mucosal Immune Responses: a microorganisms. The naturally occurring, or wild-type, Diph 60 Potential New Strategy to Target Recombinant Vaccine Anti theria toxin may be obtained from toxin producing strains gens to Mucosal Immune Systems.” Infection and Immunity available from a variety of public sources including the 73(9):5654-5665; Price, et al., 2005, “Intranasal Administra American Type Culture Collection. A plasmid system for tion of Recombinant Neisseria gonorrhoeae Transferrin producing CRM197 protein in C. diphtheriae is described by, Binding Proteins A and B Conjugated to the Cholera Toxin B e.g., U.S. Pat. No. 5,614,382, “Plasmid for Production of 65 Subunit Induces Systemic and Vaginal Antibodies in Mice.” CRM Protein and Diphtheria toxin, incorporated herein by Infection and Immunity 73(7):3945-3953; and Sun, et al., reference in its entirety. 1999, "Intranasal Administration of a Schistosoma mansoni US 8,906,636 B2 13 14 Glutathione S-Transferase-Cholera Toxoid Conjugate Vac ture-Activity Analysis of the Activation of Pertussis Toxin.” cine Evokes Antiparasitic and Antipathological Immunity in Biochemistry 26(1): 123-7; all incorporated by reference Mice.” J. Immunol. 163:1045-1052, all incorporated herein herein in their entirety. by reference. Pertussis Toxin or PTX as used herein refers to Pertussis In embodiments of the present invention, CTB or CTX is Toxin mutant S1 R9K E129A or the wild-type protein. Wild produced using any of the host strains described herein in type Pertussis toxin and Pertussis toxin mutant S1 R9K Example 1, in combination with any of the expression vectors E129A are described by, e.g.: Roberts, et al., 1995 (cited described in Example 3. In embodiments, the nucleic acid above); U.S. Pat. No. 7.427.404 and U.S. Pat. No. 7,666.436, sequence is optimized for expression in the Pseudomonad both titled, “Pertussis Toxin Mutants, Bordetella Strains host cell. In embodiments, the expression vectors used con 10 Capable of Producing Such Mutants and Their Use in the tain constructs expressing any of the secretion leaders Development of Antipertussis Vaccines: U.S. Pat. No. 5,935, described in Table 8 and Table 3 fused to the recombinant 580, “Recombinant Mutants for Inducing Specific Immune CTB or CTX protein. In embodiments, the native secretion Responses.” U.S. Pat. No. 7,169,399, “Non-Toxic Double leader is used. In certain embodiments, the CTB or CTX 15 Mutant Forms of Pertussis Toxin as Adjuvants: U.S. Pat. No. protein is expressed with a tag, e.g., a purification tag. In 5,785,971 and U.S. Pat. No. 5,427,788, both titled, “Pertussis embodiments, the methods of the invention are used to pro Toxin and Use in Vaccines; and U.S. Pat. No. 5,773,600, duce CTB or CTX at a yield of about 0.2 g/L to at least about “DNA Encoding Pertussis Toxin Muteins, all incorporated 5 g/L. herein by reference in their entirety. Pertussis Toxin In embodiments of the present invention, Pertussis toxin Pertussis toxin is an exotoxin and virulence factor pro mutant S1 E129A or wild-type Pertussis toxin is produced duced by Bordetella pertussis, a bacterial pathogen of the using any of the host strains described herein in Example 1, 5 human respiratory tract that causes the disease whooping and 7. In embodiments, the expression vectors used contain cough. The pertussis holotoxin is a multi-subunit complex constructs expressing any of the Secretion leaders described with an AB 5 structure. The enzymatically active A subunit 25 in Table 8 and Table 3 fused to the recombinant PTX protein. (S1) is an ADP-ribosyltransferase that modifies the alpha In embodiments, the native secretion leader is used. In subunit of several heterotrimeric G proteins (primarily G i embodiments, any or all of the subunit encoding sequences proteins) in mammalian cells, and the Boligomer (S2, S3, 2 are optimized for expression in the Pseudomonad host copies of S4, and S5) binds glycoconjugate receptors on cells. selected, as described elsewhere herein. In certain embodi S1 is proteolytically processed after cell entry. Carbonetti, et 30 ments, the subunits are expressed from two or more con al., 2005, “Proteolytic Cleavage of Pertussis Toxin S1 Sub structs, for example, by Subcloning the individual sequences unit is Not Essential for Its Activity in Mammalian Cells.” according to methods well-known in the art. In certain BMC Microbiology 5:7, incorporated herein by reference, embodiments, the PTX protein is expressed with a tag, e.g., a reported that processing of S1 is not essential for its cytotoxic purification tag. In embodiments, the methods of the inven activity in mammalian cells. 35 tion are used to produce PTX or each individual subunit of Nontoxic variants of Pertussis toxin have been explored for PTX at a yield of about 0.2 g/L to at least about 5 g/L. use in vaccines. Pertussis toxin protein produced using the Tetanus Toxin Fragment C methods of the present invention is contemplated for use in a Tetanus Toxin, produced by Clostridium tetani, is a neuro vaccine to protect against pertussis. Pertussis toxin has also toxin having a molecular weight of 150 kDa. It is made up of been tested as a vaccine adjuvant, e.g., as described by Rob 40 two parts: a 100 kDa heavy or B-chain and a 50 kDa light or erts, et al., 1995, “A Mutant Pertussis Toxin Molecule That A-chain. The chains are connected by a disulfide bond. The Lacks ADP-Ribosyltransferase Activity, PT-9K/129G. Is an B-chain binds to disialogangliosides (GD2 and GD1b) on the Effective Mucosal Adjuvant for Intranasally Delivered Pro neuronal membrane. The A-chain, a Zinc endopeptidase, teins.” Infection and Immunity 63(6):2100-2108, incorpo attacks the vesicle-associated membrane protein (VAMP). rated herein by reference. Further, Pertussis toxin is also 45 The action of the A-chain stops the affected neurons from useful for research purposes, e.g., for studies of G protein releasing the inhibitory neurotransmitters GABA (gamma signaling pathways (e.g., McCoy, et al., 2010, “PAR1 and aminobutyric acid) and glycine by degrading the protein Syn PAR2 couple to overlapping and distinct sets of G proteins aptobrevin. The consequence of this is dangerous overactivity and linked signaling pathways to differentially regulate cell in the muscles from the smallest stimulus—the failure of physiology. Molecular Pharmacology Fast Forward MOL 50 inhibition of motor reflexes by sensory stimulation. This 62018, incorporated herein by reference) and as an adjuvant causes generalized contractions of the agonistandantagonist to enhance induction of autoimmune diseases, e.g., experi musculature, termed a tetanic spasm. mental autoimmune encephalomyelitis (EAE), experimental Tetanus Toxin Fragment C (Tet C or TTC) is a 50 kD autoimmune orchitis, experimental autoimmune uveitis, etc. polypeptide generated by protease cleavage (e.g., with (Su, et al., 2001, “Pertussis Toxin Inhibits Induction of Tis 55 papain) of Tetanus toxin, or through recombinant expression sue-Specific Autoimmune Disease by Disrupting G Protein of the fragment. It corresponds to the 451 amino acids at the Coupled Signals. J Immunol 167:250-256incorporated C-terminus (amino acid positions 865-1315). Recombinant herein by reference). expression of Fragment C is disclosed in, e.g., U.S. Pat. No. The five subunits of the toxin are expressed from the Per 5,443.966, "Expression of Tetanus Toxin Fragment C. tussis Toxoid operon, shown in FIG. 5. The expression and 60 WO/2005/000346, “Carrier Proteins for Vaccines, and structure of Pertussis toxin proteins, including certain vari 6,010.871, “Modification of Peptide and Protein all incor ants, are described by above-cited reports, as well as by porated herein by reference in their entirety. Burnette, et al., 1992, “Properties of Pertussis Toxin B Oli Fragment Chas been shown to be non-toxic and capable of gomer Assembled In Vitro from Recombinant Polypeptides stimulating a protective immune response in mice and guinea Produced by Escherichia coli.” Infection and Immunity 65 pigs. U.S. Pat. No. 5,443,966 describes the sequence of Teta 60(6):2252-2256: U.S. Pat. No. 5,085,862, “Genetic detoxi nus Toxin and production of Fragment C in E. coli. Expres fication of pertussis toxin; and Kaslow, et al., 1987, "Struc sion of recombinant TTC in yeast has been described, e.g., in US 8,906,636 B2 15 16 U.S. Pat. No. 5,571,694, “Expression of Tetanus Toxin Frag a purification tag. In embodiments, the methods of the inven ment C in Yeast, incorporated herein by reference in its tion are used to produce TcdB at a yield of about 0.5 g/L to at entirety. least about 10 g/L. Because it binds to neurons with high specificity and affin Pseudomonas Aeruginosa Exotoxin A ity, TTC finds use as a targeting molecule for neuronal drug 5 Exotoxin A (ETA or PE) of Pseudomonas aeruginosa is a delivery or for research purposes. Such use is described by, Type II ADPRT. It is one member of a family of secreted e.g., Townsend, et al., 2007. “Tetanus toxin C fragment con bacterial toxins capable of translocating a catalytic domain jugated nanoparticles for targeted drug delivery to neurons.” into mammalian cells and inhibiting protein synthesis by the Biomaterials 28(34):5176-5184, incorporated herein by ref ADP-ribosylation of cellular elongation factor 2. The protein CCC. 10 exists as a monomer, consisting of a single polypeptide chain TTC protein is also potentially useful as a vaccine carrier of 613 amino acids (66. Kd). The X-ray crystallographic struc protein, as described in, e.g., WO/2005/000346, and has been ture of exotoxin A, determined to 3.0-A resolution, shows an explored for use in a vaccine to protect against C. tetani amino-terminal domain, composed primarily of antiparallel infection. beta-structure and comprising approximately half of the mol In embodiments of the present invention, TTC is produced 15 ecule; a middle domain composed of alpha-helices; and a using any of the host strains described herein in Example 1, in carboxyl-terminal domain comprising approximately one combination with any of the expression vectors described in third of the molecule. The carboxyl-terminal domain is the Example 8. In embodiments, the nucleic acid sequence is ADP-ribosyltransferase of the toxin. The other two domains optimized for expression in the Pseudomonad host cell. In are presumably involved in cell receptor binding and mem embodiments, the expression vectors used have constructs 20 brane translocation. expressing any of the secretion leaders described in Table 8 The toxin binds to cells through a specific receptor on the and Table 3 fused to the recombinant TTC protein. In certain cell Surface, then the toxin-receptor complex is internalized embodiments, the TTC protein is expressed with a tag, e.g., a into the cell. Finally, ETA is transferred to the cytosol where purification tag. In embodiments, the native secretion leader it enzymatically inhibits protein synthesis. The transfer pro is used. In embodiments, the methods of the invention are 25 cess is believed to occur from an acidic compartment, since used to produce TTC at a yield of about 0.5 g/L to at least cellular intoxication is prevented by weak bases such as about 12 g/L. NH4+, which raises the pH in acidic vesicles. Upon exposure C. difficile Toxin B to acidic conditions, the hydrophobic domain of PE enters Clostridium difficile Toxin B (TcdB) is a virulence factor into the membrane, resulting in the formation of a channel produced by Clostridium difficile, which causes hospital 30 through which the enzymatic domain, in extended form, acquired diarrhea and pseudomembranous colitis. TcdB, and passes into the cytosol. The activity of PE and mutants having a second large clostridial toxin, TcdA, are involved in the reduced toxicity are described in, e.g., U.S. Pat. No. 4,892, development of pseudomembranous colitis. 827, “Recombinant Pseudomonas Exotoxins: Construction TcdB, a glucosylating toxin of about 270 kD. can be of an Active Immunotoxin with Low Side Effects, and by divided into enzymatic, translocation and receptor binding 35 Lukac, et al., 1988, “Toxoid of Pseudomonas aeruginosa domains. The first 546 amino acids of TcdB contain the Exotoxin A Generated by Deletion of an Active-Site Resi enzymatic region, which is followed by a putative transloca due.” Infection and Immunity 56(12): 3095-3098, both incor tion and receptor-binding domain. Enzymatic activity has porated herein by reference in their entirety. been reported to require the amino-terminal 546 residues, as Use of Exotoxin A mutant rEPA as a vaccine conjugate is amino or carboxy-terminal deletions of this fragment 40 described by, e.g.: Fattom, et al., 1993, “Laboratory and decrease activity. Within the enzymatic region, tryptophan Clinical Evaluation of Conjugate Vaccines Composed of Sta 102 has been shown to be essential for UDP-glucose binding. phylococcus aureus Type 5 and Type 8 Capsular Polysaccha A conserved DXD motif within LCTs is essential for LCT rides Bound to Pseudomonas aeruginosa Recombinant Exo glucosyltransferase activity. Studies involving analysis of protein A.” Infection and Immunity 61(3):1023-1032; Qian, chimeras of the TcdB and TcsL enzymatic domain suggest 45 et al., 2007. “Conjugating recombinant proteins to that residues 364 to 516 confer substrate specificity. Pseudomonas aeruginosa ExoProtein A: a strategy for The structure of TcdB and its expression and potential use enhancing immunogenicity of malaria vaccine candidates.” as a protective vaccine for C. difficile infection are discussed Vaccine 25(20):3923-3933; and Lin, et al., 2001. “The Effi in, e.g.: U.S. Pat. No. 7,226,597, “Mutants of Clostridium cacy of a Salmonella Tiphi Vi Conjugate Vaccine in Two-To Difficile Toxin B and Methods of Use: Jank, et al., 2008, 50 Five-Year-Old Children.” N EnglJ Med 344(17): 1263-1269, "Structure and mode of action of clostridial glucosylating both incorporated herein by reference. toxins: the ABCD model. Trends in Microbiology 16(5): Pseudomonas aeruginosa Exotoxin Aas used herein refers 222-229; Sullivan, et al., 1982, "Purification and Character to Pseudomonas aeruginosa Exotoxin A mutant CRM66, ization of Toxins A and B of Clostridium difficile.” Infection deletion rEPA, or the wild-type protein. In embodiments of and Immunity 35(3):1032-1040; and Yang, et al., 2008, 55 the present invention, Exotoxin A is produced using any of the “Expression of recombinant Clostridium difficile toxin A and host strains described herein in Examples 1, 5 and 7, and B in Bacillus megaterium.” BMC Microbiology 8:192, all using expression vectors having constructs expressing any of incorporated herein by reference in their entirety. the secretion leaders described in Table 8 and Table 3 fused to In embodiments of the present invention, TcdB is produced the recombinant Exotoxin A protein. In embodiments, the using any of the host strains described herein in Examples 1, 60 nucleic acid sequence is optimized for expression in the 5 and 7. In embodiments, the nucleic acid sequence is opti Pseudomonad host cell. In embodiments, the native secretion mized for expression in the Pseudomonad host cell. In leader is used. In certain embodiments, the ETA protein is embodiments, the expression vectors used contain constructs expressed with a tag, e.g., a purification tag. In embodiments, expressing any of the secretion leaders described in Table 8 the methods of the invention are used to produce Exotoxin A and Table 3 fused to the recombinant TcdB protein. In 65 at a yield of about 0.5 g/L to at least about 12 g/L. embodiments, the native secretion leader is used. In certain Exemplary toxin proteins produced using the methods of embodiments, the TcdB protein is expressed with a tag, e.g., the invention are listed in Table 1. It is understood that this list US 8,906, 636 B2 17 18 is not limiting. In embodiments of the invention, any of the tiation can include a synthetic polynucleotide sequence inad nucleic acid sequences of the toxins described herein for Vertently containing motifs capable of functioning as a ribo production using the methods of the invention can be opti some binding site (RBS). These sites can result in initiating mized for expression in the Pseudomonad host cell selected. translation of a truncated protein from a gene-internal site. As described elsewhere herein, there are multiple options for 5 One method of reducing the possibility of producing a trun optimization of any given sequence. Any of the options as cated protein, which can be difficult to remove during purifi described are contemplated for use in optimizing the cation, includes eliminating putative internal RBS sequences sequences of the toxins produced using the methods of the from an optimized polynucleotide sequence. present invention. The optimized sequences provided herein Repeat-induced polymerase slippage can result in reduced are non-limiting examples of optimized sequences useful in 10 heterologous protein expression. Repeat-induced polymerase the methods of the present invention. slippage involves nucleotide sequence repeats that have been shown to cause slippage or Stuttering of DNA polymerase TABLE 1. which can result in frameshift mutations. Such repeats can also cause slippage of RNA polymerase. In an organism with Exemplary Toxin Proteins 15 a high G+C content bias, there can be a higher degree of Exemplary Sequence repeats composed of G or C nucleotide repeats. Therefore, Target Source Reference Origin one method of reducing the possibility of inducing RNA CRM197 GenBankAcc. No. Corynebacterium polymerase slippage, includes altering extended repeats of G 100721.6A diphtheriae 2O or C nucleotides. NCTC 13129 Diphtheria toxin GenBank NC OO2935.2 Corynebacterium Interfering secondary structures also can result in reduced (WT) GenBank CAAOO374.1 diphtheriae heterologous protein expression. Secondary structures can Cholera Holotoxin GenBank NC OO2505.1; Vibrio choierae sequester the RBS sequence or initiation codon and have been NP231099.1 and NP23110.1 Cholera Toxin B GenBankACH70471 Vibrio choierae O1 25 correlated to a reduction in protein expression. Stemloop (E1 Tor strain) biovar Eltor structures can also be involved in transcriptional pausing and Pertussis Toxin GenBank M13223.1 with Bordeteila pertussis attenuation. An optimized polynucleotide sequence can con mutations in S1 Tetanus Toxin C GenBank 1A8D A Cliostridium tetani tain minimal secondary structures in the RBS and gene cod Fragment ing regions of the nucleotide sequence to allow for improved C. difficile Tox B GenBank CAA63562 Clostridium difficile transcription and translation. VPI (TcdB) 30 Another feature that can effect heterologous protein Paeruginosa GenBank NP 249839 Pseudomonas Exotoxin A aeruginosa expression is the presence of restriction sites. By removing PAO1 restriction sites that could interfere with subsequent sub cloning of transcription units into host expression vectors a Codon Optimization 35 polynucleotide sequence can be optimized. In heterologous expression systems, optimization steps For example, the optimization process can begin by iden may improve the ability of the host to produce the foreign tifying the desired amino acid sequence to be heterologously protein. Protein expression is governed by a host of factors expressed by the host. From the amino acid sequence a can including those that affect transcription, mRNA processing, didate polynucleotide or DNA sequence can be designed. and stability and initiation of translation. The polynucleotide 40 During the design of the synthetic DNA sequence, the fre optimization steps may include steps to improve the ability of quency of codon usage can be compared to the codon usage of the host to produce the foreign protein as well as steps to assist the host expression organism and rare host codons can be the researcher in efficiently designing expression constructs. removed from the synthetic sequence. Additionally, the Syn Optimization strategies may include, for example, the modi thetic candidate DNA sequence can be modified in order to fication of translation initiation regions, alteration of mRNA 45 remove undesirable enzyme restriction sites and add or structural elements, and the use of different codon biases. remove any desired signal sequences, linkers or untranslated Methods for optimizing the nucleic acid sequence of to regions. The synthetic DNA sequence can be analyzed for the improve expression of a heterologous protein in a bacterial presence of secondary structure that may interfere with the host are known in the art and described in the literature. For translation process, such as G/C repeats and stem-loop struc example, optimization of codons for expression in a 50 tures. Before the candidate DNA sequence is synthesized, the Pseudomonas host strain is described, e.g., in U.S. Pat. App. optimized sequence design can be checked to Verify that the Pub. No. 2007/0292918, “Codon Optimization Method.” sequence correctly encodes the desired amino acid sequence. incorporated herein by reference in its entirety. Finally, the candidate DNA sequence can be synthesized Optimization canthus address any of a number of sequence using DNA synthesis techniques, such as those known in the features of the heterologous gene. As a specific example, a 55 art. rare codon-induced translational pause can result in reduced In another embodiment of the invention, the general codon heterologous protein expression. A rare codon-induced trans usage in a host organism, such as P. fluorescens, can be lational pause includes the presence of codons in the poly utilized to optimize the expression of the heterologous poly nucleotide of interest that are rarely used in the host organism nucleotide sequence. The percentage and distribution of may have a negative effect on protein translation due to their 60 codons that rarely would be considered as preferred for a scarcity in the available tRNA pool. One method of improv particular amino acid in the host expression system can be ing optimal translation in the host organism includes per evaluated. Values of 5% and 10% usage can be used as cutoff forming codon optimization which can result in rare host values for the determination of rare codons. For example, the codons being removed from the synthetic polynucleotide codons listed in Table 2 have a calculated occurrence of less Sequence. 65 than 5% in the Pfluorescens MB214 genome and would be Alternate translational initiation also can result in reduced generally avoided in an optimized gene expressed in a P heterologous protein expression. Alternate translational ini fluorescens host. US 8,906,636 B2 19 20 TABLE 2 TABLE 3 Codons occurring at less than 5% in Pfluorescens MB214 Exemplary Secretion Leader Sequences Amino Acid (s) Codon(s) Used % Occurrence Secretion SEQ Leader Amino Acid Sequence ID NO : G Gly GGA 3.26 IIle ATA 3.05 DsbA. RNLILSAALWTASLFGMTAOA 3 L. Leu CTA 1.78 Azul FAKLVAVSLLTLASGOLLA 4. CTT 4.57 TTA 1.89 10 Ibp - S31A IRDNRLKTSLLRGLTITLLSLTLLSP 5 R Arg AGA 1.39 AAHA AGG 2.72 CGA 4.99 Tpr NRSSALLLAFWFLSGCQAMA 6 S Ser TCT 4.28 CupB2 FRTLLASLTFAWIAGLPSTAHA 7 15 The present invention contemplates the use of any coding Cup.A2 SCTRAFKPLLLIGLATLMCSHAFA 8 sequence for the toxins produced, including any sequence NikA RIAALPLLLAPLFIAPMAWA 9 that has been optimized for expression in the Pseudomonas host cell being used. Sequences contemplated for use can be Pbp A2 OV KLKRLMAAMTFWAAGWATWNAVA O optimized to any degree as desired, including, but not limited DSC RLTOIIAAAAIALWSTFALA 1. to, optimization to eliminate: codons occurring at less than 5% in the Pseudomonas host cell, codons occurring at less ToB RNLLRGMLWWICCMAGIAAA. 2 than 10% in the Pseudomonas host cell, a rare codon-induced Pbp KLKRLMAAMTFWAAGWATANAVA 3 translational pause, a putative internal RBS sequence, an extended repeat of G or C nucleotides, an interfering second 25 LaC) QNYKKFLLAAAVSMAFSATAMA 4. ary structure, a restriction site, or combinations thereof. Cup C2 PPRSIAACLGLLGLLMATOAAA 5 Furthermore, the amino acid sequence of any secretion leader useful in practicing the methods of the present inven Port KKSTLAVAVTLGAIAQOAGA 6 tion can be encoded by any appropriate nucleic acid 30 Pbp KLKRLMAAMTFWAAGWATANAVA 7 Sequence. Expression Systems Flg.I KFKQLMAMALLLALSAVAQA 8 ttg2C QNRTVEIGWGLFLLAGILALLLLALR 9 Methods for expressing heterologous proteins, including WSGLSA useful regulatory sequences (e.g., promoters, secretion lead 35 ers, and ribosome binding sites), in Pseudomonas host cells, CRM197 SRKLFASXLIGALLGIGAPPSAHA 2O as well as host cells useful in the methods of the present native leader invention, are described, e.g., in U.S. Pat. App. Pub. No. 2008/0269070 and U.S. patent application Ser. No. 12/610, It is understood that the secretion leaders useful in the 207, both titled “Method for Rapidly Screening Microbial 40 methods of the present invention are not limited to those Hosts to Identify Certain Strains with Improved Yield and/or disclosed in Table 3. Quality in the Expression of Heterologous Proteins. U.S. In embodiments, the secretion leader is AZu, IbpS31A, Pat. App. Pub. No. 2006/0040352, “Expression of Mamma Cup A2, or Pbp A20V. In other embodiments, the secretion lian Proteins in Pseudomonas Fluorescens, and U.S. Pat. leader is Azu, IbpS31A, Cup A2, PbpA20V, or Pbp. App. Pub. No. 2006/0110747, “Process for Improved Protein 45 Native CRM197 is transported from C. diptheriae to the Expression by Strain Engineering, all incorporated herein by extracellular space via a secretion leader that is cleaved, leav reference in their entirety. These publications also describe ing an amino terminal sequence of GADD (SEQID NO: 21). bacterial host strains useful in practicing the methods of the In order to preserve the natural amino terminus of CRM197 invention, that have been engineered to overexpress folding following expression in Pfluorescens and ensure disulfide modulators or wherein protease mutations, including dele 50 bond formation, the protein is targeted to the periplasmic tions, have been introduced, in order to increase heterologous Space. protein expression. Promoters The promoters used in accordance with the present inven Leaders tion may be constitutive promoters or regulated promoters. Sequence leaders are described in detail in U.S. Patent 55 Common examples of useful regulated promoters include App. Pub. Nos. 2008/0193974 and 2010/0048864, both titled, those of the family derived from the lac promoter (i.e. the lacz “Bacterial Leader Sequences for Increased Expression.” and promoter), especially the tac and trc promoters described in U.S. Pat. App. Pub. No. 2006/0008877, “Expression systems U.S. Pat. No. 4,551,433 to DeBoer, as well as Ptac16, Ptac17, with Sec-secretion, all incorporated herein by reference in PtacII, PlacUV5, and the T71ac promoter. In one embodi 60 ment, the promoter is not derived from the host cell organism. their entirety, as well as in U.S. Pat. App. Pub. No. 2008/ In certain embodiments, the promoter is derived from an E. 0269070 and U.S. patent application Ser. No. 12/610,207. coli organism. In embodiments, a sequence encoding a secretion leader is Inducible promoter sequences can be used to regulate fused to the sequence encoding the toxin protein. In embodi expression of the toxins inaccordance with the methods of the ments, the secretion leader is a periplasmic secretion leader. 65 invention. In embodiments, inducible promoters useful in the In embodiments, the secretion leader is the native secretion methods of the present invention include those of the family leader. derived from the lac promoter (i.e. the lacZ promoter), espe US 8,906,636 B2 21 22 cially the tac and trc promoters described in U.S. Pat. No. of host cell biomass, an appropriate effector compound is 4,551,433 to DeBoer, as well as Ptac16, Ptac17, PtacII, added to the culture to directly or indirectly result in expres PlacUV5, and the T71ac promoter. In one embodiment, the sion of the desired gene(s) encoding the protein or polypep promoter is not derived from the host cell organism. In certain tide of interest. embodiments, the promoter is derived from an E. coli organ 5 In embodiments wherein a lac family promoter is utilized, ism. a lad gene can also be present in the system. The lad gene, Common examples of non-lac-type promoters useful in which is normally a constitutively expressed gene, encodes expression systems according to the present invention the Lac repressor protein Lad protein, which binds to the lac include, e.g., those listed in Table 4. operator of lac family promoters. Thus, where a lac family 10 promoter is utilized, the lad gene can also be included and expressed in the expression system. TABLE 4 Promoter systems useful in Pseudomonas are described in Examples of non-lac Promoters the literature, e.g., in U.S. Pat. App. Pub. No. 2008/0269070, also referenced above. Promoter Inducer 15 Other Regulatory Elements PR High temperature In embodiments, soluble proteins are present in either the P. High temperature cytoplasm or periplasm of the cell during production. Secre Pn Alkyl- or halo-benzoates tion leaders useful for targeting proteins are described else Pl Alkyl- or halo-toluenes Psal Salicylates where herein, and in U.S. Pat. App. Pub. No. 2008/0193974, U.S. Pat. App. Pub. No. 2006/0008877, and in U.S. patent application Ser. No. 12/610,207. See, e.g.: J. Sanchez-Romero & V. De Lorenzo (1999) Other elements include, but are not limited to, transcrip Manual of Industrial Microbiology and Biotechnology (A. tional enhancer sequences, translational enhancer sequences, Demain & J. Davies, eds.) pp. 460-74 (ASM Press, Washing other promoters, activators, translational start and stop sig ton, D.C.); H. Schweizer (2001) Current Opinion in Biotech 25 nals, transcription terminators, cistronic regulators, polycis nology, 12:439-445; and R. Slater & R. Williams (2000 tronic regulators, tag sequences, such as nucleotide sequence Molecular Biology and Biotechnology (J. Walker & R. Rap "tags' and “tag” polypeptide coding sequences, which facili ley, eds.) pp. 125-54 (The Royal Society of Chemistry, Cam tates identification, separation, purification, and/or isolation bridge, UK)). A promoter having the nucleotide sequence of of an expressed polypeptide. a promoter native to the selected bacterial host cell also may 30 In embodiments, the expression vector further comprises a be used to control expression of the transgene encoding the tag sequence adjacent to the coding sequence for the secretion target polypeptide, e.g., a Pseudomonas anthranilate or ben signal or to the coding sequence for the protein or polypeptide Zoate operon promoter (Pant, Pben). Tandem promoters may of interest. In one embodiment, this tag sequence allows for also be used in which more than one promoter is covalently purification of the protein. The tag sequence can be an affinity attached to another, whether the same or different in 35 tag, such as a hexa-histidine affinity tag (SEQID NO: 46). In sequence, e.g., a Pant-Pben tandem promoter (interpromoter another embodiment, the affinity tag can be a glutathione-S- hybrid) or a Plac-Plac tandem promoter, or whether derived transferase molecule. The tag can also be a fluorescent mol from the same or different organisms. ecule, such as YFP or GFP, or analogs of such fluorescent Regulated promoters utilize promoter regulatory proteins proteins. The tag can also be a portion of an antibody mol in order to control transcription of the gene of which the 40 ecule, or a known antigen or ligand for a known binding promoter is a part. Where a regulated promoter is used herein, partner useful for purification. a corresponding promoter regulatory protein will also be part An expression construct useful in practicing the methods of an expression system according to the present invention. of the present invention can include, in addition to the protein Examples of promoter regulatory proteins include: activator coding sequence, the following regulatory elements operably proteins, e.g., E. coli catabolite activator protein, MalT pro 45 linked thereto: a promoter, a ribosome binding site (RBS), a tein; AraC family transcriptional activators; repressor pro transcription terminator, and translational start and stop sig teins, e.g., E. coli Lad proteins; and dual-function regulatory nals. Useful RBSs can be obtained from any of the species proteins, e.g., E. coli NagO protein. Many regulated-pro useful as host cells in expression systems according to, e.g., moter/promoter-regulatory-protein pairs are known in the art. U.S. Pat. App. Pub. No. 2008/0269070 and U.S. patent appli In one embodiment, the expression construct for the target 50 cation Ser. No. 12/610,207. Many specific and a variety of protein(s) and the heterologous protein of interest are under consensus RBSS are known, e.g., those described in and ref the control of the same regulatory element. erenced by D. Frishman et al., Gene 234(2):257-65 (8 Jul. Promoter regulatory proteins interact with an effector com 1999); and B. E. Suzek et al., Bioinformatics 17(12): 1123-30 pound, i.e., a compound that reversibly or irreversibly asso (December 2001). In addition, either native or synthetic RBSs ciates with the regulatory protein so as to enable the protein to 55 may be used, e.g., those described in: EP 0207459 (synthetic either release or bind to at least one DNA transcription regu RBSs); O. Ikehata et al., Eur. J. Biochem. 181(3):563-70 latory region of the gene that is under the control of the (1989) (native RBS sequence of AAGGAAG). Further promoter, thereby permitting or blocking the action of a tran examples of methods, vectors, and translation and transcrip Scriptase enzyme in initiating transcription of the gene. Effec tion elements, and other elements useful in the present inven tor compounds are classified as either inducers or co-repres 60 tion are well known in the art and described in, e.g.: U.S. Pat. sors, and these compounds include native effector No. 5,055.294 to Gilroy and U.S. Pat. No. 5,128,130 to Gilroy compounds and gratuitous inducer compounds. Many regu et al.; U.S. Pat. No. 5,281,532 to Rammler et al.; U.S. Pat. lated-promoter/promoter-regulatory-protein/effector-com Nos. 4,695,455 and 4,861,595 to Barnes et al.; U.S. Pat. No. pound trios are known in the art. Although an effector com 4.755,465 to Gray et al.; and U.S. Pat. No. 5,169,760 to pound can be used throughout the cell culture or 65 Wilcox, all incorporated herein by reference, as well as in fermentation, in a preferred embodiment in which a regulated many of the other publications incorporated herein by refer promoter is used, after growth of a desired quantity or density CCC. US 8,906,636 B2 23 24 Host Strains TABLE 5-continued Bacterial hosts, including Pseudomonads, and closely related bacterial organisms are contemplated for use in prac Families and Genera Listed in the Part, “Gram-Negative Aerobic Rods ticing the methods of the invention. In certain embodiments, and Cocci” (Bergey, 1974) the Pseudomonad host cell is Pseudomonas fluorescens. The Family II. Azotobacteraceae Azomonas Azotobacter host cell can also be an E. coli cell. Beijerinckia Host cells and constructs useful in practicing the methods Dexia of the invention can be identified or made using reagents and Family III. Rhizobiaceae Agrobacterium Rhizobium methods known in the art and described in the literature, e.g., 10 Family IV. Methylomonadaceae Methylococcus in U.S. Pat. App. Pub. No. 2009/0325230, “Protein Expres Methylomonas sion Systems, incorporated herein by reference in its Family V. Halobacteriaceae Haiobacterium Haiococcus entirety. This publication describes production of a recombi Other Genera Acetobacter nant polypeptide by introduction of a nucleic acid construct Alcaligenes into an auxotrophic Pseudomonas fluorescens host cell com 15 Bordeteia Bruceiia prising a chromosomal lad gene insert. The nucleic acid con Franciselia struct comprises a nucleotide sequence encoding the recom Thermits binant polypeptide operably linked to a promoter capable of directing expression of the nucleic acid in the host cell, and also comprises a nucleotide sequence encoding an aux Pseudomonas and closely related bacteria are generally otrophic selection marker. The auxotrophic selection marker part of the group defined as “Gram(-) Proteobacteria Sub is a polypeptide that restores prototrophy to the auxotrophic group 1 or “Gram-Negative Aerobic Rods and Cocci' host cell. In embodiments, the cell is auxotrophic for proline, (Buchanan and Gibbons (eds.) (1974) Bergey’s Manual of uracil, or combinations thereof. In embodiments, the host cell Determinative Bacteriology, pp. 217-289). Pseudomonas is derived from MB101 (ATCC deposit PTA-7841). U.S. Pat. 25 host strains are described in the literature, e.g., in U.S. Pat. App. Pub. No. 2009/0325230, "Protein Expression Systems.” App. Pub. No. 2006/0040352, cited above. and in Schneider, et al., 2005, 'Auxotrophic markers pyrF and “Gram-Negative Proteobacteria Subgroup 1 also includes proC can replace antibiotic markers on protein production Proteobacteria that would be classified in this heading plasmids in high-cell-density Pseudomonas fluorescens fer 30 according to the criteria used in the classification. The head mentation. Biotechnol. Progress 21(2): 343-8, both incorpo ing also includes groups that were previously classified in this rated herein by reference in their entirety, describe a produc Section but are no longer, Such as the genera Acidovorax, tion host strain auxotrophic for uracil that was constructed by Brevundimonas, Burkholderia, Hydrogenophaga, Oceani deleting the pyrE gene in strain MB101. The pyrF gene was monas, Ralstonia, and Stenotrophomonas, the genus Sphin cloned from strain MB214 (ATCC deposit PTA-7840) to generate a plasmid that can complement the pyrF deletion to 35 gomonas (and the genus Blastomonas, derived therefrom), restore prototropy. In particular embodiments, a dual pyrF which was created by regrouping organisms belonging to proC dual auxotrophic selection marker system in a Pfluo (and previously called species of) the genus Xanthomonas, rescens host cell is used. A PyrE production host strain as the genus Acidomonas, which was created by regrouping described can be used as the background for introducing other organisms belonging to the genus Acetobacter as defined in desired genomic changes, including those described hereinas 40 Bergey (1974). In addition hosts can include cells from the useful in practicing the methods of the invention. genus Pseudomonas, Pseudomonas enalia (ATCC 14393), In embodiments, the host cell is of the order Pseudomonad Pseudomonas nigrifaciensi (ATCC 19375), and Pseudomo ales. Where the host cell is of the order Pseudomonadales, it nas putrefaciens (ATCC 8071), which have been reclassified may be a member of the family Pseudomonadaceae, includ 45 respectively as Alteromonas haloplanktis, Alteromonas nigri ing the genus Pseudomonas. Gamma Proteobacterial hosts include members of the species Escherichia coli and mem faciens, and Alteromonas putrefaciens. Similarly, e.g., bers of the species Pseudomonas fluorescens. Pseudomonas acidovorans (ATCC 15668) and Pseudomonas Other Pseudomonas organisms may also be useful. testosteroni (ATCC 1 1996) have since been reclassified as Pseudomonads and closely related species include Gram Comamonas acidovorans and Comamonas testosteroni, negative Proteobacteria Subgroup 1, which include the group 50 respectively; and Pseudomonas nigrifaciens (ATCC 19375) of Proteobacteria belonging to the families and/or genera and Pseudomonas piscicida (ATCC 15057) have been reclas described as “Gram-Negative Aerobic Rods and Cocci' by R. sified respectively as Pseudoalteromonas nigrifaciens and E. Buchanan and N. E. Gibbons (eds.), Bergey’s Manual of Pseudoalteromonas piscicida. “Gram-negative Proteobacte Determinative Bacteriology, pp. 217-289 (8th ed., 1974) (The ria Subgroup 1 also includes Proteobacteria classified as Williams & Wilkins Co., Baltimore, Md., USA) (hereinafter “Bergey (1974)). Table 5 presents these families and genera belonging to any of the families: Pseudomonadaceae, AZoto of organisms. bacteraceae (now often called by the synonym, the Azoto bacter group' of Pseudomonadaceae), Rhizobiaceae, and TABLE 5 Methylomonadaceae (now often called by the synonym, 60 “Methylococcaceae). Consequently, in addition to those Families and Genera Listed in the Part, “Gram-Negative Aerobic Rods genera otherwise described herein, further Proteobacterial and Cocci” (Bergey, 1974) genera falling within “Gram-negative Proteobacteria Sub Family I. Pseudomonaceae Gluconobacter group 1 include: 1) Azotobacter group bacteria of the genus Pseudomonas Xanthomonas 65 Azorhizophilus; 2) Pseudomonadaceae family bacteria of the Zoogloea genera Cellvibrio, Oligella, and Teredinibacter; 3) Rhizobi aceae family bacteria of the genera Chelatobacter, Ensifer, US 8,906,636 B2 25 26 Liberibacter (also called “Candidatus Liberibacter'), and Stutzeri (ATCC 17588); Pseudomonas amygdali (ATCC Sinorhizobium; and 4) Methylococcaceae family bacteria of 33614); Pseudomonas avellanae (ATCC 700331); the genera Methylobacter, Methylocaldum, Methylomicro Pseudomonas caricapapayae (ATCC 33615); Pseudomonas bium, Methylosarcina, and Methylosphaera. cichorii (ATCC 10857); Pseudomonas ficuserectae (ATCC The host cell can be selected from “Gram-negative Proteo 35104); Pseudomonas fiscovaginae, Pseudomonas meliae bacteria Subgroup 16.” “Gram-negative Proteobacteria Sub (ATCC 33050); Pseudomonas syringae (ATCC 19310): group 16' is defined as the group of Proteobacteria of the Pseudomonas viridiflava (ATCC 13223); Pseudomonas ther following Pseudomonas species (with the ATCC or other mocarboxydovorans (ATCC 35961); Pseudomonas thermo deposit numbers of exemplary strain(s) shown in parenthe 10 tolerans, Pseudomonas thivervalensis, Pseudomonas van sis): Pseudomonas abietaniphila (ATCC 700689); Pseudomonas aeruginosa (ATCC 101.45); Pseudomonas couverensis (ATCC 700688); Pseudomonas wisconsinensis; alcaligenes (ATCC 14909); Pseudomonas anguilliseptica and Pseudomonas xiamenensis. In one embodiment, the host (ATCC 33660); Pseudomonas citronellolis (ATCC 13674): cell is Pseudomonas fluorescens. 15 The host cell can also be selected from “Gram-negative Pseudomonas flavescens (ATCC 51555); Pseudomonas men Proteobacteria Subgroup 17.” “Gram-negative Proteobacte docina (ATCC 25411); Pseudomonas nitroreducens (ATCC ria Subgroup 17 is defined as the group of Proteobacteria 33634); Pseudomonas oleovorans (ATCC 8062); Pseudomo known in the art as the “fluorescent Pseudomonads' includ nas pseudoalcaligenes (ATCC 17440); Pseudomonas resino ing those belonging, e.g., to the following Pseudomonas spe vorans (ATCC 14235); Pseudomonas straminea (ATCC cies: Pseudomonas azotoformans, Pseudomonas brenneri; 33.636); Pseudomonas agarici (ATCC 25941); Pseudomonas Pseudomonas cedrella, Pseudomonas COrrugata, alcaliphila, Pseudomonas alginovora, Pseudomonas ander Pseudomonas extremorientalis, Pseudomonas fluorescens, sonii. Pseudomonas asplenii (ATCC 23835); Pseudomonas Pseudomonas gessardii. Pseudomonas libanensis, azelaica (ATCC 27162): Pseudomonas beverinckii (ATCC Pseudomonas mandelii, Pseudomonas marginalis, 19372); Pseudomonas borealis, Pseudomonas boreopolis 25 Pseudomonas migulae, Pseudomonas mucidolens, (ATCC 33662); Pseudomonas brassicacearum, Pseudomo Pseudomonas Orientalis, Pseudomonas rhodesiae, Pseudomonas synxantha, Pseudomonas tolaasii; and nas butanovora (ATCC 43655); Pseudomonas cellulosa Pseudomonas veronii. (ATCC 55703); Pseudomonas aurantiaca (ATCC 33663); In embodiments, the Pseudomonas host cell is defective in Pseudomonas chlororaphis (ATCC 9446, ATCC 13985, 30 the expression of HslU, HslV. Prc1, DegP1, DegP2, AprA, or ATCC 17418, ATCC 17461); Pseudomonas fragi (ATCC a combination thereof. In embodiments, the host cell is defec 4973); Pseudomonas lundensis (ATCC 49968); Pseudomo tive in proteases HslU, HsV. Prel, DegP1, DegP2, and AprA, nas taetrolens (ATCC 4683); Pseudomonascissicola (ATCC and overexpresses DegP2S219A. An example of such a strain 33616); Pseudomonas coronafaciens, Pseudomonas diter is disclosed herein as Host Strain 2. These proteases are peniphila, Pseudomonas elongata (ATCC 10144); 35 known in the art and described in, e.g., U.S. Pat. App. Pub. No. Pseudomonasflectens (ATCC 12775); Pseudomonas azoto 2006/01 10747. AprA, an extracellular serralysin-type metal formans, Pseudomonas brenneri. Pseudomonas cedrella, loprotease metalloproteinase, is described by, e.g., Maunsell, Pseudomonas corrugata (ATCC 29736); Pseudomonas et al., 2006, “Complex regulation of AprA metalloprotease in Pseudomonas fluorescens M114: evidence for the involve extremorientalis, Pseudomonas fluorescens (ATCC 35858); 40 ment of iron, the ECF sigma factor, PbrA and pseudobactin Pseudomonas gessardii. Pseudomonas libanensis, M114 siderophore, Microbiology 152(Pt 1):29-42, incorpo Pseudomonas mandelii (ATCC 700871); Pseudomonas mar rated herein by reference, and in U.S. Patent App. Pub. Nos. ginalis (ATCC 10844); Pseudomonas migulae, Pseudomo 2008/O193974 and 2010/0048864. nas mucidolens (ATCC 4685); Pseudomonas orientalis, In other embodiments, the Pseudomonas host cell overex Pseudomonas rhodesiae, Pseudomonas synxantha (ATCC 45 presses DsbA, DsbB, DsbC, and DsbD. DsbA, B, C, and D 9890); Pseudomonas tolaasii (ATCC 33618); Pseudomonas are disulfide bond isomerases, described, e.g., in U.S. Pat. veronii (ATCC 700474); Pseudomonas federiksbergensis, App. Pub. No. 2008/0269070 and U.S. patent application Ser. Pseudomonas geniculata (ATCC 19374); Pseudomonas gin No. 12/610,207. geri. Pseudomonas graminis, Pseudomonas grimontii, In other embodiments, the Pseudomonas host cell is wild 50 type, i.e., having no protease expression defects and not over Pseudomonas halodenitrificans, Pseudomonas halophila, expressing any folding modulator. Pseudomonas hibiscicola (ATCC 19867); Pseudomonas hut A host cell that is defective in the expression of a protease tiensis (ATCC 14670); Pseudomonas hydrogenovora, can have any modification that results in a decrease in the Pseudomonas jessenii (ATCC 700870); Pseudomonas kilon normal activity or expression level of that protease relative to ensis, Pseudomonas lanceolata (ATCC 14669); Pseudomo 55 a wild-type host. For example, a missense or nonsense muta nas lini. Pseudomonas marginate (ATCC 25417); tion can lead to expression of protein that not active, and a Pseudomonas mephitica (ATCC 33665); Pseudomonas deni gene deletion can result in no protein expression at all. A trificans (ATCC 19244); Pseudomonas pertucinogena change in the upstream regulatory region of the gene can (ATCC 190); Pseudomonas pictorum (ATCC 23328); result in reduced or no protein expression. Other gene defects 60 can affect translation of the protein. The expression of a Pseudomonas psychrophila, Pseudomonas filva (ATCC protease can also be defective if the activity of a protein 31418); Pseudomonas monteilii (ATCC 700476); Pseudomo needed for processing the protease is defective. nas mosselii, Pseudomonas oryzihabitans (ATCC 43272): Examples of proteases andfolding modulators useful in the Pseudomonas plecoglossicida (ATCC 700383); Pseudomo methods of the present invention are shown in Tables 6 and 7. nas putida (ATCC 12633); Pseudomonas reactans, 65 respectively. RXF numbers refer to the open reading frame. Pseudomonas spinosa (ATCC 14606); Pseudomonas bale (See, e.g., U.S. Pat. App. Pub. No. 2008/0269070 and U.S. arica, Pseudomonas luteola (ATCC 43273); Pseudomonas patent application Ser. No. 12/610,207.) US 8,906,636 B2 27 28 TABLE 6 A fluorescens Strain MB214 proteases Class Family RXF Gene Curated Function Location Aspartic Peptidases A8 (signal peptidase II family) RXFOS383.2 Lipoprotein signal peptidase (ec Cytoplasmic 3.4.23.36) Membrane A24 (type IV prepilin peptidase family) RXFO5379.1 type 4 prepilin peptidase pild (ec Cytoplasmic 3.4.99.—) Membrane Cysteine Peptidases C15 (pyroglutamyl peptidase I family) RXFO2.161.1 Pyrrollidone-carboxylate Cytoplasmic peptidase (ec 3.4.19.3) C40 RXFO1968.1 invasion-associated protein, P60 Signal peptide RXFO4920.1 invasion-associated protein, P60 Cytoplasmic RXFO4923.1 phosphatase-associated protein Signal peptide papq C56 (PfpI endopeptidase family) RXFO1816.1 protease I (ec 3.4.——) Non-secretory Metallopeptidases M1 RXFO8773.1 Membrane alanine Non-secretory aminopeptidase (ec 3.4.11.2) M3 RXFOO561.2 prlC Oligopeptidase A (ec 3.4.24.70) Cytoplasmic RXFO4631.2 Zn-dependent oligopeptidases Cytoplasmic M4 (thermolysin family) RXFOS113.2 Extracellular metalloprotease Extracellular precursor (ec 3.4.24.—) M41 (FtsHendopeptidase family) RXFOS4OO.2 Cell division protein ftsH (ec Cytoplasmic 3.4.24.—) Membrane M10 RXFO43O4.1 Serralysin (ec 3.4.24.40) Extracellular RXFO4SOO.1 Serralysin (ec 3.4.24.40) Extracellular RXFO1590.2 Serralysin (ec 3.4.24.40) Extracellular RXFO4497.2 Serralysin (ec 3.4.24.40) Extracellular RXFO4495.2 Serralysin (ec 3.4.24.40) Extracellular RXFO2796.1 Serralysin (ec 3.4.24.40) Extracellular M14 (carboxypeptidase A family) RXFO9091.1 Zinc-carboxypeptidase precursor Cytoplasmic (ec 3.4.17.—) M16 (pitrilysin family) RXFO3441.1 Coenzyme pad synthesis protein Non-secretory F (ec 3.4.99.—) RXFO1918.1 Zinc protease (ec 3.4.99.—) Signal peptide RXFO1919.1 Zinc protease (ec 3.4.99.—) Periplasmic RXFO3699.2 processing peptidase (ec Signal peptide 3.4.24.64) M17 (leucyl aminopeptidase family) RXFOO285.2 Cytosol aminopeptidase (ec Non-secretory 3.4.11.1) M18 RXFO7879.1 Aspartyl aminopeptidase (ec Cytoplasmic 3.4.11.21) M2O RXFOO811.1 dapF. Succinyl-diaminopimelate Cytoplasmic desuccinylase (ec 3.5.1.18) RXFO4052.2 Xaa-His dipeptidase (ec Signal peptide 3.4.13.3) RXFO1822.2 Carboxypeptidase G2 precursor Signal peptide (ec 3.4.17.11) RXFO9831:2:: N-acyl-L-amino acid Signal peptide RXFO4892.1 amidohydrolase (ec 3.5.1.14) M28 (aminopeptidase Y family) RXFO3488.2 Alkaline phosphatase isozyme OuterMembrane conversion protein precursor (ec 3.4.11.—) M42 (glutamylaminopeptidase family) RXFO5615.1 Deblocking aminopeptidase (ec Non-secretory 3.4.11.—) M22 RXFOS817.1 O-Sialoglycoprotein Extracellular endopeptidase (ec 3.4.24.57) RXFO3065.2 Glycoprotease protein family Non-secretory M23 RXFO1291.2 Cell wall endopeptidase, family Signal peptide M23, M37 RXFO3916. Membrane proteins related to Signal peptide metalloendopeptidases RXFO9147.2 Cell wall endopeptidase, family Signal peptide M23, M37 M24 RXFO4693. Methionine aminopeptidase (ec Cytoplasmic 3.4.11.18) RXFO3364. Methionine aminopeptidase (ec Non-secretory 3.4.11.18) RXFO2980. Xaa-Pro aminopeptidase (ec Cytoplasmic 3.4.11.9) RXFO6564. Xaa-Pro aminopeptidase (ec Cytoplasmic 3.4.11.9) M48 (Ste24 endopeptidase family) RXFO5137. Heat shock protein HtpX Cytoplasmic Membrane RXFOSO81. Zinc metalloprotease (ec 3.4.24.—) Signal peptide M50 (S2P protease family) RXFO4692. Membrane metalloprotease Cytoplasmic Membrane US 8,906,636 B2 29 30 TABLE 6-continued A fluorescens Strain MB214 proteases Class Family RXF Gene Curated Function Location Serine Peptidases S1 (chymotrypsin family) RXFO1250.2 protease do (ec 3.4.21.—) Periplasmic RXFO7210.1 protease do (ec 3.4.21.—) Periplasmic S8 (subtilisin family) RXFO6755.2 serine protease (ec 3.4.21.—) Non-secretory RXFO8517.1 serine protease (ec 3.4.21.—) Extracellular RXFO8627.2 extracellular serine protease (ec Signal peptide 3.4.21.—) RXFO6281.1 Extracellular serine protease Non-secretory precursor (ec 3.4.21.—) RXFO8978.1 extracellular serine protease (ec OuterMembrane 3.4.21.—) RXFO6451.1 serine protease (ec 3.4.21.—) Signal peptide S9 (prolyl oligopeptidase family) RXFO2OO3.2 Protease ii (ec 3.4.21.83) Periplasmic RXFOO458.2 Hydrolase Non-secretory S11 (D-Ala-D-Ala carboxypeptidase RXFO4657.2 D-alanyl-D-alanine- Periplasmic A family) endopeptidase (ec 3.4.99.—) RXFOO670.1 D-alanyl-D-alanine Cytoplasmic carboxypeptidase (ec 3.4.16.4) Membrane S13 (D-Ala-D-Ala peptidase C family) RXFOO133.1 D-alanyl-meso-diaminopimelate OuterMembrane endopeptidase (ec 3.4.—.—) RXFO4960.2 D-alanyl-meso-diaminopimelate Signal peptide endopeptidase (ec 3.4.—.—) S14 (ClpP endopeptidase family) RXFO4567.1 clpP atp-dependent Clp protease Non-secretory proteolytic subunit (ec 3.4.21.92) RXFO4663.1 clpP atp-dependent Clp protease Cytoplasmic proteolytic subunit (ec 3.4.21.92) S16 (lon protease family) RXFO4653.2 atp-dependent protease La (ec Cytoplasmic 3.4.21.53) RXFO8653. atp-dependent protease La (ec Cytoplasmic 3.4.21.53) RXFOS943. atp-dependent protease La (ec Cytoplasmic 3.4.21.53) S24 (LexA family) RXFOO449. LexA repressor (ec 3.4.21.88) Non-secretory RXFO3397. LexA repressor (ec 3.4.21.88) Cytoplasmic S26 (signal peptidase I family) RXFO1181. Signal peptidase I (ec 3.4.21.89) Cytoplasmic Membrane S33 RXFOS236. pip3 Proline iminopeptidase (ec Non-secretory 3.4.11.5) RXFO48O2. pip1 Proline iminopeptidase (ec Non-secretory 3.4.11.5) RXFO4808.2 pip2 Proline iminopeptidase (ec Cytoplasmic 3.4.11.5) S41 (C-terminal processing peptidase RXFO6586 Tail-specific protease (ec Signal peptide family) 3.4.21.—) RXFO1037 Tail-specific protease (ec Signal peptide 3.4.21.—) S45 RXFO7170 pacB2 Penicillin acylase (ec 3.5.1.11) Signal peptide RXFO 6399.2 pacB1 Penicillin acylase ii (ec 3.5.1.11) Signal peptide S49 (protease IV family) RXFO6993.2 possible protease Sohb (ec 3.4.——) Non-Secretory RXFO1418. protease iv (ec 3.4.——) Non-secretory S58 (Dmp A aminopeptidase family) RXFO63O8.2 D-aminopeptidase (ec 3.4.11.19) Cytoplasmic Membrane Threonine Peptidases T1 (proteasome family) RXFO1961.2 hSIV atp-dependent protease hslV (ec Cytoplasmic 3.4.25.—) T3 (gamma-glutamyltransferase family) RXFO2342. ggt1 Gamma-glutamyltranspeptidase Periplasmic (ec 2.3.2.2) RXFO44242 ggt2 Gamma-glutamyltranspeptidase Periplasmic (ec 2.3.2.2) Unclassified Peptidases U32 RXFOO428 protease (ec 3.4.——) Cytoplasmic RXFO2151.2 protease (ec 3.4.——) Cytoplasmic U61 RXFO4715 Muramoyltetrapeptide Non-secretory carboxypeptidase (ec 3.4.17.13) U62 RXFO4971.2 pmbA PmbA protein Cytoplasmic RXFO4968.2 TldD protein Cytoplasmic Non MEROPS Proteases RXFOO325 Repressor protein C2 Non-secretory RXFO2689.2 Microsomal dipeptidase (ec Cytoplasmic 3.4.13.19) RXFO2739 membrane dipeptidase Signal peptide (3.4.13.19) RXFO3329.2 Hypothetical Cytosolic Protein Cytoplasmic RXFO2492 Xaa-Pro dipeptidase (ec Cytoplasmic 3.4.13.9) US 8,906,636 B2 31 32 TABLE 6-continued A fluorescens Strain MB214 proteases Class Family RXF Gene Curated Function Location RXFO4047.2 caax amino terminal protease Cytoplasmic family Membrane RXFO8136.2 protease (transglutaminase-like Cytoplasmic protein) RXFO9487.1 Zinc metalloprotease (ec 3.4.24.—) Non-secretory
Certain proteases can have both protease and chaperone RXFO1961.2 (hslV); Peptidyl-prolyl cis-trans isomerase like activity. When these proteases are negatively affecting family member RXF05345.2 (ppiB); Metallopeptidase M20 protein yield and/or quality it can be useful to delete them, and they can be overexpressed when their chaperone activity 15 family member RXF04892.1 (aminohydrolase); Metallopep may positively affect protein yield and/or quality. These pro tidase M24 family members RXFO4693.1 (methionine ami teases include, but are not limited to: Hsp100 (Clp/Hsl) fam nopeptidase) and RXFO3364.1 (methionine aminopepti ily members RXF04587.1 (clpA), RXF08347.1, dase); and Serine Peptidase S26 signal peptidase I family RXF04654.2 (clpX), RXFO4663.1, RXFO1957.2 (hslU), member RXFO1181.1 (signal peptidase). TABLE 7 A fluorescens strain MB214 protein folding modulators
ORFID GENE FUNCTION FAMILY LOCATION GroESEL RXFO2095.1 groES Chaperone Hsp10 Cytoplasmic RXFO6767.1: groEL Chaperone Hsp60 Cytoplasmic Rxf)2O90 RXFO1748.1 ibpA Small heat-shock protein (SEISP) Ibp A HS2O Cytoplasmic PA3126; Acts as a holder for GroESL folding RXFO3385.1 hscB Chaperone protein hscE HSb2O Cytoplasmic Hsp70 (DnaK/J) RXFOS399.1 dnaK Chaperone Hsp70 Periplasmic RXFO69S4.1 dnaK Chaperone Hsp70 Cytoplasmic RXFO3376.1 hscA Chaperone Hsp70 Cytoplasmic RXFO3987.2 cbp.A Curved dina-binding protein, dinal like Hsp40 Cytoplasmic activity RXFOS4O6.2 dna Chaperone protein dna Hsp40 Cytoplasmic RXFO3346.2 dna Molecular chaperones (DnaJ family) Hsp40 Non-secretory RXFOS413.1 grpE heat shock protein GrpE PA4762 GrpE Cytoplasmic Hsp100 (Clp/Hsl) RXFO4587.1 clip A atp-dependent clp protease atp-binding Hsp100 Cytoplasmic Subunit clip A RXFO8347.1 clipB ClpB protein Hsp100 Cytoplasmic RXFO4654.2 clpX atp-dependent clp protease atp-binding Hsp100 Cytoplasmic subunit clpX RXFO4663.1 clpP atp-dependent Clp protease proteolytic MEROPS Cytoplasmic subunit (ec 3.4.21.92) peptidase amily S14 RXFO 1957.2 hisU atp-dependent his protease atp-binding Hsp100 Cytoplasmic subunithsU RXFO 1961.2 hSV atp-dependent his protease proteolytic MEROPS Cytoplasmic subunit peptidase subfamily T1B Hsp33 RXFO4254.2 yrfI 33 kDa chaperonin (Heat shock protein Hsp33 Cytoplasmic 33 homolog) (HSP33). Hsp90 RXFOS455.2 htpG Chaperone protein htpG Hsp90 Cytoplasmic SecB
RXFO2231.1 SecB secretion specific chaperone SecB SecB Non-secretory Disulfide Bond Isomerases
RXFO7017.2 disbA disulfide isomerase DSBA oxido- Cytoplasmic reductase US 8,906,636 B2 33 34 TABLE 7-continued A fluorescens Strain MB214 protein folding modulators
ORFID GENE FUNCTION FAMILY LOCATION RXFO8657.2 disbA disulfide isomerase DSBA oxido- Cytoplasmic dsbC reductase dsbGif fernA RXFO1 OO2.1 disbA disulfide isomerase DSBA oxido- Periplasmic dsbC reductase? Thioredoxin RXFO3307.1 dsbC disulfide isomerase Glutaredoxin Periplasmic Thioredoxin RXFO4890.2 dsbG disulfide isomerase Glutaredoxin Periplasmic Thioredoxin RXFO32O4.1 dsbB Disulfide bond formation protein B DSBA oxido- Periplasmic (Disulfide oxidoreductase). reductase RXFO4886.2 dsbD Thiol:disulfide interchange protein dsbD DSBA oxido- Periplasmic reductase Peptidyl-prolyl cis-trans isomerases RXFO3768.1 ppiA Peptidyl-prolyl cis-trans isomerase A (ec PPIase: Periplasmic 5.2.1.8) cyclophilin type RXFOS345.2 ppiB Peptidyl-prolyl cis-trans isomerase B. PPIase: Cytoplasmic cyclophilin type RXFO6034.2 kIB Peptidyl-prolyl cis-trans isomerase FklB. PPIase: OuterMembrane FKBP type RXFO6591.1 kBf k506 binding protein Peptidyl-prolyl cis- PPIase: Periplasmic kbP trans isomerase (EC 5.2.1.8) FKBP type RXFO5753.2 kIB; Peptidyl-prolyl cis-trans isomerase (ec PPIase: Outer kbP 5.2.1.8) FKBP type Membrane RXFO1833.2 slyD Peptidyl-prolyl cis-trans isomerase Sly D. PPIase: Non-secretory FKBP type RXFO4655.2 ig Trigger factor, ppiase (ec 5.2.1.8) PPIase: Cytoplasmic FKBP type RXFOS385. yaad Probable FKBP-type 16 kDa peptidyl- PPIase: Non-secretory prolyl cis-trans isomerase (EC 5.2.1.8) FKBP type (PPiase) (Rotamase). RXFOO271. Peptidyl-prolyl cis-trans isomerase (ec PPIase: Non-secretory 5.2.1.8) FKBP type pili assembly chaperones (papD like)
RXFO6068. Cl Chaperone protein cup pili assembly Periplasmic papD RXFO5719. ecpD Chaperone protein ecpD pili assembly Signal peptide papD RXFOS319. ecpD Hnr protein pili assembly Periplasmic chaperone RXFO34O6.2 ecpD: Chaperone protein ecpD pili assembly Signal peptide cSuC papD RXFO4296. ecpD: Chaperone protein ecpD pili assembly Periplasmic Cl papD RXFO4S53. ecpD: Chaperone protein ecpD pili assembly Periplasmic Cl papD RXFO4554.2 ecpD: Chaperone protein ecpD pili assembly Periplasmic Cl papD RXFOS31 O.2 ecpD: Chaperone protein ecpD pili assembly Periplasmic Cl papD RXFOS3O4.1 ecpD: Chaperone protein ecpD pili assembly Periplasmic Cl papD RXFO5073.1 gltF Gram-negative pili assembly chaperone pili assembly Signal peptide periplasmic function papD Type II Secretion Complex RXFOS445.1 Yac Histidinol-phosphate aminotransferase (ec Class-II Membrane 2.6.1.9) pyridoxal phosphate dependent aminotransferase amily. Histidinol phosphate aminotransferase Subfamily. US 8,906,636 B2 35 36 TABLE 7-continued A fluorescens Strain MB214 protein folding modulators
ORFID GENE FUNCTION FAMILY LOCATION RXFOS426.1 SecD Protein translocase subunit seco Type II Membrane Secretion complex RXFOS432.1 SecF protein translocase subunit sec? Type II Membrane Secretion complex Disulfide Bond Reductases
RXFO8122.2 trixC Thioredoxin 2 Disulfide Cytoplasmic Bond Reductase RXFO6751.1 Gor Glutathione reductase (EC 1.8.1.7) (GR) Disulfide Cytoplasmic (GRase) PA2025 Bond Reductase RXFOO922.1 gshA Glutamate-cysteine ligase (ec 6.3.2.2) Disulfide Cytoplasmic PAS2O3 Bond Reductase
High Throughput Screens erologous proteins are described, for example, in U.S. Patent In some embodiments, a high throughput screen can be Application Publication No. 20080269070. conducted to determine optimal conditions for expressing a Fermentation Format soluble recombinant toxin protein. The conditions that be 25 The expression system according to the present invention varied in the screen include, for example, the host cell, genetic can be cultured in any fermentation format. For example, background of the host cell (e.g., deletions of different pro batch, fed-batch, semi-continuous, and continuous fermenta teases), type of promoter in an expression construct, type of tion modes may be employed herein. secretion leader fused to the sequence encoding the recom In embodiments, the fermentation medium may be 30 selected from among rich media, minimal media, and mineral binant protein, growth temperature, OD at induction when an salts media. In other embodiments either a minimal medium inducible promoter is used, concentration of IPTG used for or a mineral salts medium is selected. In certain embodi induction when a lacZ promoter is used, duration of protein ments, a mineral salts medium is selected. induction, growth temperature following addition of an Mineral salts media consists of mineral salts and a carbon inducing agent to a culture, rate of agitation of culture, 35 Source Such as, e.g., glucose, Sucrose, or glycerol. Examples method of selection for plasmid maintenance, Volume of cul of mineral salts media include, e.g., M9 medium, Pseudomo ture in a vessel, and method of cell lysing. nas medium (ATCC 179), and Davis and Mingioli medium In some embodiments, a library (or "array') of host strains (see, BD Davis & ESMingioli (1950).J. Bact. 60:17-28). The is provided, wherein each strain (or “population of host mineral salts used to make mineral salts media include those cells') in the library has been genetically modified to modu 40 selected from among, e.g., potassium phosphates, ammo late the expression of one or more target genes in the host cell. nium Sulfate or chloride, magnesium Sulfate or chloride, and An “optimal host strain” or “optimal expression system can trace minerals such as calcium chloride, borate, and Sulfates be identified or selected based on the quantity, quality, and/or of iron, copper, manganese, and Zinc. Typically, no organic location of the expressed protein of interest compared to other nitrogen source. Such as peptone, tryptone, amino acids, or a populations of phenotypically distinct host cells in the array. 45 yeast extract, is included in a mineral salts medium. Instead, Thus, an optimal host strain is the strain that produces the an inorganic nitrogen Source is used and this may be selected polypeptide of interest according to a desired specification. from among, e.g., ammonium salts, aqueous ammonia, and While the desired specification will vary depending on the gaseous ammonia. A mineral salts medium will typically polypeptide being produced, the specification includes the contain glucose or glycerol as the carbon Source. In compari quality and/or quantity of protein, e.g., whether the protein is 50 son to mineral salts media, minimal media can also contain sequestered or secreted, and in what quantities, whether the mineral salts and a carbon Source, but can be supplemented protein is properly or desirably processed and/or folded, and with, e.g., low levels of amino acids, vitamins, peptones, or the like. In embodiments, improved or desirable quality can other ingredients, though these are added at very minimal be production of toxin protein with high fidelity cleavage of levels. Media can be prepared using the methods described in the secretion leader and low levels of degradation. In embodi 55 the art, e.g., in U.S. Pat. App. Pub. No. 2006/0040352, refer ments, the optimal host strain or optimal expression system enced and incorporated by reference above. Details of culti produces a yield, characterized by the amount or quantity of Vation procedures and mineral salts media useful in the meth soluble heterologous protein, the amount or quantity of ods of the present invention are described by Riesenberg, Det recoverable heterologous protein, the amount or quantity of al., 1991, “High cell density cultivation of Escherichia coli at properly processed heterologous protein, the amount or quan 60 controlled specific growth rate. J. Biotechnol. 20 (1):17-27. tity of properly folded heterologous protein, the amount or In embodiments, production can be achieved in bioreactor quantity of active heterologous protein, and/or the total cultures. Cultures can be grown in, e.g., up to 2 liter bioreac amount or quantity of heterologous protein, of a certain abso tors containing a mineral salts medium, and maintained at 32° lute level or a certain level relative to that produced by an C. and pH 6.5 through the addition of ammonia. Dissolved indicator Strain, i.e., a strain used for comparison. 65 oxygen can be maintained in excess through increases in Methods of screening microbial hosts to identify strains agitation and flow of sparged air and oxygen into the fermen with improved yield and/or quality in the expression of het tor. Glycerol can be delivered to the culture throughout the US 8,906,636 B2 37 38 fermentation to maintain excess levels. In embodiments, 29°C., about 30°C., about 31°C., about 32°C., about 33°C., these conditions are maintained until a target culture cell about 34°C., about 35° C., about 36°C., about 37°C., about density, e.g., optical density at 575 nm (A575), for induction 38°C., about 39°C., about 40°C., about 41° C., or about 42° is reached, at which time IPTG is added to initiate the target C. In other embodiments, the growth temperature is main protein production. It is understood that the cell density at 5 tained at about 25°C. to about 27°C., about 25°C. to about induction, the concentration of IPTG, pH and temperature 28°C., about 25°C. to about 29°C., about 25°C. to about 30° each can be varied to determine optimal conditions for C., about 25°C. to about 31°C., about 25°C. to about 32°C., expression. In embodiments, cell density at induction can be about 25° C. to about 33°C., about 26° C. to about 28°C., varied from A575 of 40 to 200 absorbance units (AU). IPTG concentrations can be varied in the range from 0.02 to 1.0 10 about 26° C. to about 29° C., about 26° C. to about 30° C., mM, pH from 6 to 7.5, and temperature from 20 to 35° C. about 26° C. to about 31° C., about 26° C. to about 32° C., After 16-24 hours, the culture from each bioreactor can be about 27°C. to about 29° C., about 27°C. to about 30° C., harvested by centrifugation and the cell pellet frozen at -80° about 27°C. to about 31° C., about 27°C. to about 32° C., C. Samples can then be analyzed, e.g., by SDS-CGE, for about 26° C. to about 33°C., about 28°C. to about 30° C., product formation. 15 about 28°C. to about 31° C., about 28°C. to about 32° C., Fermentation may be performed at any scale. The expres about 29° C. to about 31° C., about 29° C. to about 32° C., sion systems according to the present invention are useful for about 29° C. to about 33°C., about 30° C. to about 32° C., recombinant protein expression at any scale. Thus, e.g., about 30° C. to about 33°C., about 31° C. to about 33°C., microliter-scale, milliliter scale, centiliter scale, and deciliter about 31° C. to about 32°C., about 30° C. to about 33°C., or scale fermentation Volumes may be used, and 1 Liter scale 20 about 32° C. to about 33° C. In other embodiments, the and larger fermentation Volumes can be used. temperature is changed during culturing. In one embodiment, In embodiments, the fermentation volume is at or above the temperature is maintained at about 30° C. before an agent about 1 Liter. In embodiments, the fermentation volume is to induce expression from the construct, e.g., IPTG, is added about 1 liter to about 100 liters. In embodiments, the fermen to the culture. After adding the induction agent, the tempera tation volume is about 1 liter, about 2 liters, about 3 liters, 25 ture is reduced to about 25°C. about 4 liters, about 5 liters, about 6 liters, about 7 liters, about Induction 8 liters, about 9 liters, or about 10 liters. In embodiments, the As described elsewhere herein, inducible promoters can be fermentation volume is about 1 liter to about 5 liters, about 1 used in the expression construct to control expression of the liter to about 10 liters, about 1 liter to about 25 liters, about 1 recombinant toxin protein, e.g., a lac promoter. In the case of liter to about 50 liters, about 1 liter to about 75 liters, about 10 30 the lac promoter derivatives or family members, e.g., the tac liters to about 25 liters, about 25 liters to about 50 liters, or promoter, the effector compound is an inducer, such as a about 50 liters to about 100 liters. In other embodiments, the gratuitous inducer like IPTG (isopropyl-B-D-1-thiogalacto fermentation volume is at or above 5 Liters, 10 Liters, 15 pyranoside, also called "isopropylthiogalactoside'). In Liters, 20 Liters, 25 Liters, 50 Liters, 75 Liters, 100 Liters, embodiments, a lac promoter derivative is used, and recom 200 Liters, 500 Liters, 1,000 Liters, 2,000 Liters, 5,000 35 binant protein expression is induced by the addition of IPTG Liters, 10,000 Liters, or 50,000 Liters. to a final concentration of about 0.01 mM to about 1.0 mM, Bacterial Growth Conditions when the cell density has reached a level identified by an Growth conditions useful in the methods of the provided OD575 of about 80 to about 160. In embodiments, the OD575 invention can comprise a temperature of about 4°C. to about at the time of culture induction for the recombinant protein 42°C. and a pH of about 5.7 to about 8.8. When an expression 40 can be about 80, about 90, about 100, about 110, about 120, construct with a lacZ promoter is used, expression can be about 130, about 140, about 150, about 160, about 170 about induced by adding IPTG to a culture at a final concentration of 180. In other embodiments, the OD575 is about 80 to about about 0.01 mM to about 1.0 mM. 100, about 100 to about 120, about 120 to about 140, about The pH of the culture can be maintained using pH buffers 140 to about 160. In other embodiments, the OD575 is about and methods known to those of skill in the art. Control of pH 45 80 to about 120, about 100 to about 140, or about 120 to about during culturing also can be achieved using aqueous ammo 160. In other embodiments, the OD575 is about 80 to about nia. In embodiments, the pH of the culture is about 5.7 to 140, or about 100 to 160. The cell density can be measured by about 8.8. In certain embodiments, the pH is about 5.7. 5.8, other methods and expressed in other units, e.g., in cells per 5.9, 6.0, 6.1, 6.2, 6.3, 6.4., 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, unit volume. For example, an OD575 of about 80 to about 160 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 50 of a Pseudomonas fluorescens culture is equivalent to 8.7, or 8.8 In other embodiments, the pH is about 5.7 to 5.9, approximately 8x1010 to about 1.6x1011 colony forming 5.8 to 6.0, 5.9 to 6.1, 6.0 to 6.2, 6.1 to 6.3, 6.2 to 6.5, 6.4 to 6.7, units per mL or 35 to 70 g/L dry cell weight. In embodiments, 6.5 to 6.8, 6.6 to 6.9, 6.7 to 7.0, 6.8 to 7.1, 6.9 to 7.2, 7.0 to 7.3, the cell density at the time of culture induction is equivalent to 7.1 to 7.4, 7.2 to 7.5, 7.3 to 7.6, 7.4 to 7.7, 7.5 to 7.8, 7.6 to 7.9, the cell density as specified herein by the absorbance at 7.7 to 8.0, 7.8 to 8.1, 7.9 to 8.2, 8.0 to 8.3, 8.1 to 8.4, 8.2 to 8.5, 55 OD575, regardless of the method used for determining cell 8.3 to 8.6, 8.4 to 8.7, or 8.5 to 8.8. In yet other embodiments, density or the units of measurement. One of skill in the art will the pH is about 5.7 to 6.0, 5.8 to 6.1, 5.9 to 6.2, 6.0 to 6.3, 6.1 know how to make the appropriate conversion for any cell to 6.4, or 6.2 to 6.5. In certain embodiments, the pH is about culture. 5.7 to about 6.25. In embodiments, the final IPTG concentration of the cul In embodiments, the growth temperature is maintained at 60 ture is about 0.01 mM, about 0.02 mM, about 0.03 mM, about about 4° C. to about 42° C. In certain embodiments, the 0.04 mM, about 0.05 mM, about 0.06 mM, about 0.07 mM, growth temperature is about 4°C., about 5°C., about 6°C., about 0.08 mM, about 0.09 mM, about 0.1 mM, about 0.2 about 7°C., about 8°C., about 9°C., about 10°C., about 11° mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 C., about 12° C., about 13°C., about 14° C., about 15° C., mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, or about 1 about 16°C., about 17°C., about 18°C., about 19°C., about 65 mM. In other embodiments, the final IPTG concentration of 20°C., about 21°C., about 22°C., about 23°C., about 24°C., the culture is about 0.08 mM to about 0.1 mM, about 0.1 mM about 25°C., about 26°C., about 27°C., about 28°C., about to about 0.2 mM, about 0.2 mM to about 0.3 mM, about 0.3 US 8,906,636 B2 39 40 mM to about 0.4 mM, about 0.2 mM to about 0.4 mM, about Useful measures of protein yield include, e.g., the amount 0.08 to about 0.2 mM, or about 0.1 to 1 mM. of recombinant protein per culture Volume (e.g., grams or In embodiments wherein a non-lac type promoter is used, milligrams of protein/liter of culture), percent or fraction of as described herein and in the literature, other inducers or recombinant protein measured in the insoluble pellet effectors can be used. In one embodiment, the promoter is a obtained after cell lysis (e.g., amount of recombinant protein constitutive promoter. in extract Supernatant/amount of protein in insoluble frac After adding and inducing agent, cultures can be grown for tion), percent or fraction of active protein (e.g., amount of a period of time, for example about 24 hours, during which active proteinfamount protein used in the assay), percent or time the recombinant protein is expressed. After adding an fraction of total cell protein (tcp), amount of protein/cell, and inducing agent, a culture can be grown for about 1 hr, about 2 10 percent or proportion of dry biomass. In embodiments, the hr, about 3 hr, about 4 hr, about 5 hr, about 6 hr, about 7 hr, measure of protein yield as described herein is based on the about 8 hr, about 9 hr, about 10 hr, about 11 hr., about 12 hr. amount of soluble protein or the amount of active protein, or about 13 hr, about 14 hr, about 15 hr, about 16 hr, about 17 hr, both, obtained. about 18 hr, about 19 hr, about 20 hr, about 21 hr., about 22 hr. In embodiments wherein yield is expressed in terms of about 23 hr, about 24 hr, about 36 hr, or about 48 hr. After an 15 culture volume the culture cell density may be taken into inducing agent is added to a culture, the culture can be grown account, particularly when yields between different cultures for about 1 to 48 hrs, about 1 to 24 hrs, about 10 to 24 hrs, are being compared. about 15 to 24 hrs, or about 20 to 24 hrs. Cell cultures can be In embodiments, the methods of the present invention can concentrated by centrifugation, and the culture pellet resus be used to obtain a soluble and/or active and/or properly pended in a buffer or Solution appropriate for the Subsequent processed (e.g., having the Secretion leader cleaved properly) lysis procedure. recombinant toxin protein or subunit protein yield of about In embodiments, cells are disrupted using equipment for 0.2 grams per liter to about 12 grams per liter. In embodi high pressure mechanical cell disruption (which are available ments, the yield is about 0.5 grams per liter to about 12 grams commercially, e.g., Microfluidics Microfluidizer, Constant per liter. In certain embodiments, the recombinant protein or Cell Disruptor, Niro-Soavi homogenizer or APV-Gaulin 25 subunit protein yield is about 0.2 g/L, about 0.3 g/L, about 0.4 homogenizer). Cells expressing the recombinant protein can g/L, about 0.5g/L, about 0.6g/L, about 0.7 g/L, about 0.8 g/L, be disrupted, for example, using Sonication. Any appropriate about 0.9 g/L, about 1 g/L, about 1.5 g/L, about 2 g/L, about method known in the art for lysing cells can be used to release 2.5g/L, about 3 g/L, about 3.5g/L, about 4 g/L, about 4.5g/L, the soluble fraction. For example, in embodiments, chemical about 5g/L, about 5.5g/L, about 6 g/L, about 6.5 g/L, about and/or enzymatic cell lysis reagents, such as cell-wall lytic 30 7 g/L, about 7.5g/L, about 8 g/L, about 8.5g/L, about 9 g/L, enzyme and EDTA, can be used. Use of frozen or previously about 9.5 g/L, about 10 g/L, about 10.5 g/L, about 11 g/L, stored cultures is also contemplated in the methods of the about 12 g/L, about 0.2 g/L to about 0.5g/L, about 0.2 g/L to invention. Cultures can be OD-normalized prior to lysis. For about 1 g/L, about 0.2 to about 2 g/L, about 0.3 g/L to about example, cells can be normalized to an OD600 of about 10, 0.6g/L, about 0.3 g/L to about 1 g/L, about 0.3 to about 2 g/L, about 11, about 12, about 13, about 14, about 15, about 16, 35 about 0.4 to about 0.7 g/L, about 0.4 to about 1 g/L about 0.4 about 17, about 18, about 19, or about 20. to about 2 g/L, about 0.4 to about 3 g/L, about 0.5g/L to about Centrifugation can be performed using any appropriate 1 g/L, about 0.5 g/L to about 1 g/L, about 0.5 g/L to about 2 equipment and method. Centrifugation of cell culture or g/L, about 0.5 g/L to about 3 g/L, about 0.5 g/L to about 4 g/L, lysate for the purposes of separating a soluble fraction from about 0.5 g/L to about 5g/L, about 0.5 g/L to about 6 g/L, an insoluble fraction is well-known in the art. For example, 40 about 0.5 g/L to about 7 g/L, about 0.5 g/L to about 8 g/L, lysed cells can be centrifuged at 20,800xg for 20 minutes (at about 0.5 g/L to about 9 g/L, about 0.5 g/L to about 10 g/L, 4°C.), and the Supernatants removed using manual or auto about 0.5 g/L to about 11 g/L, about 0.5 g/L to about 12 g/L, mated liquid handling. The pellet (insoluble) fraction is resus about 1 g/L to about 2 g/L, about 1 g/L to about 3 g/L, about pended in a buffered solution, e.g., phosphate buffered saline 1 g/L to about 4 g/L, about 1 g/L to about 5 g/L, about 1 g/L (PBS), pH 7.4. Resuspension can be carried out using, e.g., 45 to about 6 g/L, about 1 g/L to about 7 g/L, about 1 g/L to about equipment Such as impellers connected to an overhead mixer, 8 g/L, about 1 g/L to about 9 g/L, about 1 g/L to about 10 g/L. magnetic stir-bars, rocking shakers, etc. about 1 g/L to about 11 g/L, about 1 g/L to about 12 g/L, about A “soluble fraction, i.e., the soluble supernatant obtained 2 g/L to about 3 g/L, about 2 g/L to about 4 g/L, about 2 g/L after centrifugation of a lysate, and an “insoluble fraction.” to about 5g/L, about 2 g/L to about 6 g/L, about 2 g/L to about i.e., the pellet obtained after centrifugation of a lysate, result 50 7 g/L, about 2 g/L to about 8 g/L, about 2 g/L to about 9 g/L. from lysing and centrifuging the cultures. These two fractions about 2 g/L to about 10 g/L, about 2 g/L to about 11 g/L, about also can be referred to as a “first soluble fraction' and a “first 2 g/L to about 12 g/L, about 3 g/L to about 4 g/L, about 3 g/L insoluble fraction.” respectively. to about 5g/L, about 3 g/L to about 6 g/L, about 3 g/L to about Evaluation of Product 7 g/L, about 3 g/L to about 8 g/L, about 3 g/L to about 9 g/L, Numerous assay methods are known in the art for charac 55 about 3 g/L to about 10 g/L, about 3 g/L to about 11 g/L, about terizing proteins. Use of any appropriate method for charac 3 g/L to about 12 g/L, about 4 g/L to about 5g/L, about 4 g/L terizing the yield or quality of the recombinant toxin protein to about 6 g/L, about 4 g/L to about 7 g/L, about 4 g/L to about is contemplated herein. 8 g/L, about 4 g/L to about 9 g/L, about 4 g/L to about 10 g/L. Protein Yield about 4 g/L to about 11 g/L, about 4 g/L to about 12 g/L, about Protein yield in any purification fraction as described 60 5 g/L to about 6 g/L, about 5 g/L to about 7 g/L, about 5 g/L herein can be determined by methods known to those of skill to about 8 g/L, about 5 g/L to about 9 g/L, about 5 g/L to about in the art, for example, by capillary gel electrophoresis 10 g/L, about 5 g/L to about 11 g/L, about 5 g/L to about 12 (CGE), and Western blot analysis. Activity assays, as g/L, about 6 g/L to about 7 g/L, about 6 g/L to about 8 g/L. described herein and known in the art, also can provide infor about 6 g/L to about 9 g/L, about 6 g/L to about 10 g/L, about mation regarding protein yield. In embodiments, these or any 65 6 g/L to about 11 g/L, about 6 g/L to about 12 g/L, about 7 g/L other methods known in the art are used to evaluate proper to about 8 g/L, about 7 g/L to about 9 g/L, about 7 g/L to about processing of a protein, e.g., proper secretion leader cleavage. 10 g/L, about 7 g/L to about 11 g/L, about 7 g/L to about 12 US 8,906,636 B2 41 42 g/L, about 8 g/L to about 9 g/L, about 8 g/L to about 10 g/L, active, or both soluble and active, by methods known to those about 8 g/L to about 11 g/L, about 8 g/L to about 12 g/L, about of skill in the art and described herein. The “activity” of a 9 g/L to about 10 g/L, about 9 g/L to about 11 g/L, about 9 g/L given protein can include binding activity, e.g., that repre to about 12 g/L, about 10 g/L to about 11 g/L, about 10 g/L to sented by binding to a receptor, a specific antibody, or to about 12 g/L, or about 11 g/L to about 12 g/L. 5 another known Substrate, or by enzymatic activity if relevant. In embodiments, the amount of recombinant toxin protein Activity levels can be described, e.g., in absolute terms or in or subunit protein produced is about 1% to 75% of the total relative terms, as when compared with the activity of a stan cell protein. In certain embodiments, the amount of toxin dard or control sample, or any sample used as a reference. protein or subunit protein produced is about 1%, about 2%, Activity assays for evaluating toxins are known in the art about 3%, about 4%, about 5%, about 10%, about 15%, about 10 and described in the literature. Activity assays include immu 20%, about 25%, about 30%, about 35%, about 40%, about nological or antibody binding assays, e.g., Western Blot 45%, about 50%, about 55%, about 60%, about 65%, about analysis and ELISA, as well as receptor binding assays, e.g., 70%, about 75%, about 1% to about 5%, about 1% to about CRM197 can be evaluated by Diptheria toxin receptor 10%, about 1% to about 20%, about 1% to about 30%, about (proHB-EGF) binding assay. Antibodies useful in these 1% to about 40%, about 1% to about 50%, about 1% to about 15 assays are commercially available. Activity assays also 60%, about 1% to about 75%, about 2% to about 5%, about include enzyme activity assays. Wild-type DT can be assayed 2% to about 10%, about 2% to about 20%, about 2% to about immunologically and also by ADP-ribosylation activity, 30%, about 2% to about 40%, about 2% to about 50%, about using methods known in the art and described elsewhere 2% to about 60%, about 2% to about 75%, about 3% to about herein for P aeruginosa Exotoxin A. 5%, about 3% to about 10%, about 3% to about 20%, about For example, Western blot analysis of CTB can be per 3% to about 30%, about 3% to about 40%, about 3% to about formed as described, e.g., in U.S. Pat. No. 6,140,082, 50%, about 3% to about 60%, about 3% to about 75%, about “Expression of Gene Products from Genetically Manipulated 4% to about 10%, about 4% to about 20%, about 4% to about Strains of Bordetella, incorporated herein by reference. This 30%, about 4% to about 40%, about 4% to about 50%, about patent describes expression of CTB in Bordetella. The pro 4% to about 60%, about 4% to about 75%, about 5% to about 25 teins from culture supernatants were resolved by SDS-PAGE 10%, about 5% to about 20%, about 5% to about 30%, about or boiled before being resolved to convert the CTB pentamer 5% to about 40%, about 5% to about 50%, about 5% to about to the monomeric form. The proteins were transferred onto 60%, about 5% to about 75%, about 10% to about 20%, about nylon membranes and probed with goat anti-choleragenoid 10% to about 30%, about 10% to about 40%, about 10% to IgG antibody (anti-CTB, List Biologicals iGAC-01C). about 50%, about 10% to about 60%, about 10% to about 30 Detection was performed with alkaline phosphatase-conju 75%, about 20% to about 30%, about 20% to about 40%, gated donkey anti-goat IgG, using dig chemiluminescence about 20% to about 50%, about 20% to about 60%, about 20% (Boehringer Mannheim). A Cholera toxin standard (Sigma) to about 75%, about 30% to about 40%, about 30% to about containing both CTA and CTB was used for comparison. 50%, about 30% to about 60%, about 30% to about 75%, Western blot analysis of PTX can be performed, e.g., as about 40% to about 50%, about 40% to about 60%, about 40% 35 described herein in the Examples, using commercially avail to about 75%, about 50% to about 60%, about 50% to about able antibodies. Monoclonal antibodies are available from, 75%, about 60% to about 75%, or about 70% to about 75%, of e.g., Abcam, Cambridge, Mass. the total cell protein. Tetanus Toxin C Fragment can be evaluated by Western In certain embodiments, multiple proteins are produced Blot analysis, or by ELISA as described in, e.g., U.S. Pat. No. from the same host cell. For example, in embodiments, all five 40 5,443.966, "Expression of tetanus toxin fragment C. incor subunits of Pertussis toxin are made from the same host cell porated herein by reference. Antibodies are available from grown in a single culture. In Such embodiments the concen multiple commercial sources, e.g., Abcam, Cambridge, tration, % total cell protein, or activity observed is that for Mass. each individual toxin subunit or for all the subunits taken TcdB activity can be evaluated by Western Blot or other together. That is, in embodiments, the methods of the inven 45 detection analysis, as described in the art. Enzymatic activity tion are used to obtain a yield of the S1, S2, S3, S4, or S5 can be assayed, e.g., using glucosylhydrolase/glucosylation subunit of Pertussis toxin protein of about 1 gram per liter to assay methods described in the art, for example in U.S. Pat. about 12 grams per liter. In embodiments, the amount of S1, No. 7,226,597, incorporated herein by reference in its S2, S3, S4, or S5 subunit protein produced is 1% to 75% of the entirety. Specifically, glucosylation reactions can be carried total cell protein. Alternatively, the methods of the invention 50 out in a reaction mix containing 50 mM n-2hydroxyethylpip are used to obtain a yield of S1, S2, S3, S4, and S5 subunit erazine-n'-2-ethane sulfonic acid, 100 mM KC1, 1 mM protein of about 1 gram per liter to about 12 grams per liter. In MnC12, 1 mM MgCl2, 100 gram/ml BSA, 0.2 mM GDP, 40 embodiments, the amount of S1, S2, S3, S4, and S5 subunit uMUDP-glucose (303 Ci/mol; ICN Pharmaceuticals), 100 protein produced is 1% to 75% of the total cell protein. In uMUDP-glucose and 3 pmol of TcdB or 10 pmol of each certain embodiments, the amount of each Subunit obtained, in 55 fusion protein. The assay is allowed to incubate overnight at grams per liter or % total cell protein, is approximately the 37° C. and the cleaved glucose is separated using AG1-X2 SaC. anion exchange resin and counted in a liquid Scintillation The “solubility” and “activity” of a protein, though related COunter. qualities, are generally determined by different means. The P. aeruginosa Exotoxin A activity can be evaluated using solubility of a protein, particularly a hydrophobic protein, 60 immunological methods, e.g., Western Blot analysis. Since typically relates to the folding of a protein; insolubility indi ETA is an ADP-ribosylating toxin, it can be assayed for cates that hydrophobic amino acid residues are improperly ADP-ribosylation activity, e.g., as described in U.S. Pat. No. located on the outside of the folded protein. Protein activity, 4,892.827, incorporated herein by reference. Specifically, which can be evaluated using methods, e.g., those described rabbit reticulocyte preparations or wheat germ extracts below, is another indicator of proper protein conformation. 65 enriched with elongation factor 2 (EF-2) are used as a source “Soluble, active, or both, or “soluble and/or active, as used of EF-2. Assays (500 ul total volume) contain about 10 pmole herein, refers to protein that is determined to be soluble, of EF-2, 37 pmole of 14C-NAD (0.06 ICi), 0.25 to 1.25ug of US 8,906,636 B2 43 44 ETA and buffer (40 mM DTT, 1 mM EDTA, and 50 mM Tris, EXAMPLES pH 8.1). Activity is measured as pmoles of NAD transferred to EF-2 in 30 minutes. A standard curve of known concentra Example 1 tions of PE is established and used to determine the activity of PE in extracts from E. coli. After incubation for 30 minutes at 5 High Throughput Expression of a Recombinant 37°C., 0.5 ml 12% TCA is added to each assay mixture. The CRM197 Protein assay mixtures are then set in an ice bath for 15 minutes, followed by centrifugation at 4°C., 3,000xg for 10 minutes. CRM197 expression strains were constructed and the The pellet is washed with 1 ml 6% TCA and centrifuged as amount of soluble CRM197 protein produced in the strains 10 was analyzed using capillary gel electrophoresis (SDS above. The pellet is then measured for 14C radioactivity in a CGE). Based on the resulting data, certain strains were liquid scintillation counter as the index of the ADP-ribosyla selected for use in large-scale expression. tion activity. Construction and Growth of CRM197 Expression Strains Therefore, a measure of activity can represent, e.g., anti The CRM197 coding sequence was constructed using P body or receptor binding capacity, Substrate binding capacity 15 fluorescens preferred codons to encode the CRM197 amino (as to a column material), or enzyme activity. acid sequence. FIG. 1 shows the amino acid and DNA In embodiments, activity is represented by the '% active sequences of the expressed synthetic CRM197 gene. recombinant toxin protein in the extract Supernatant as com Plasmids carrying the optimized CRM197 sequence, fused pared with the total amount assayed. This is based on the to ten Pfluorescens secretion leaders as shown in Table 8. amount of recombinant toxin protein determined to be active were constructed. The CRM197 coding sequence was fused by the assay relative to the total amount of recombinant toxin in frame with that of Pfluorescens secretion leaders to target protein used in the assay. In other embodiments, activity is the protein to the periplasm for recovery in the properly represented by the '% activity level of the protein compared to folded and active form. a standard, e.g., native protein. This is based on the amount of active recombinant toxin protein in Supernatant extract TABLE 8 sample relative to the amount of active protein in a standard 25 sample (where the same amount of protein from each sample Secretion leaders used for CRM197 expression screen is used in assay). In embodiments, about 40% to about 100% of the toxin Secretion Leader protein or subunit is determined to be active. In embodiments, DsbA. about 40%, about 50%, about 60%, about 70%, about 80%, 30 AZu about 90%, or about 100% of the recombinant toxin protein or Ibp-S31A Tpr protein subunit is determined to be active. In embodiments, CupB2 about 40% to about 50%, about 50% to about 60%, about 60% Cup A2 to about 70%, about 70% to about 80%, about 80% to about NikA 90%, about 90% to about 100%, about 50% to about 100%, 35 Pbp A2OV about 60% to about 100%, about 70% to about 100%, about DsbC 80% to about 100%, about 40% to about 90%, about 40% to 1 ToB about 95%, about 50% to about 90%, about 50% to about 95%, about 50% to about 100%, about 60% to about 90%, Constructs containing the ten secretion leaders fused to the about 60% to about 95%, about 60% to about 100%, about 40 recombinant CRM197 coding sequence were tested in P. 70% to about 90%, about 70% to about 95%, about 70% to fluorescens hosts. Four hosts, listed in Table 9, were tested about 100%, or about 70% to about 100% of the recombinant with each expression plasmid. Host cells were electroporated toxin protein or subunit is determined to be active. with the indicated plasmids, resuspended in HTP growth In other embodiments, about 75% to about 100% of the medium with trace minerals and 5% glycerol and then trans recombinant toxin protein or protein subunit is determined to ferred to 96-well deep well plate with 400 ul M9 salts 1% be active. In embodiments, about 75% to about 80%, about 45 glucose medium and trace elements. The 96-well plates were 75% to about 85%, about 75% to about 90%, about 75% to incubated at 30°C. with shaking for 48 hours. Ten microliters about 95%, about 80% to about 85%, about 80% to about of each of the forty seed cultures were transferred into tripli 90%, about 80% to about 95%, about 80% to about 100%, cate 96-well deep-well plates, each well containing 500 ul of about 85% to about 90%, about 85% to about 95%, about 85% HTP medium supplemented with trace elements and 5% to about 100%, about 90% to about 95%, about 90% to about 50 glycerol, and incubated as before for 24 hours. 100%, or about 95% to about 100% of the recombinant toxin protein or subunit is determined to be active. Means of confirming the identity of the induced protein are TABLE 9 also known in the art. For example, a protein can analyzed by Host strains used for CRM197 expression screen peptide mass fingerprint using MALDI-TOF mass spectrom etry, N-terminal sequencing analysis, or peptide mapping. 55 Host Strain Genotype Type While preferred embodiments of the present invention 1 lon, la, aprA PD have been shown and described herein, it will be obvious to 2 hslUV pre1 degP1 degP2 aprA deletions; PD + FMO those skilled in the art that such embodiments are provided by overexpresses DegP2 S219A way of example only. Numerous variations, changes, and 3 dsbABCD FMO substitutions will now occur to those skilled in the art without 60 4 grpE, dinaKJ FMO departing from the invention. It should be understood that PD = Protease Deletion (listed proteases are deleted); various alternatives to the embodiments of the invention FMO = Folding Modulator Overexpressor (listed folding modulators are overexpressed. described herein may be employed in practicing the inven tion. It is intended that the following claims define the scope Isopropyl-B-D-1-thiogalactopyranoside (IPTG) was of the invention and that methods and structures within the 65 added to each well to a final concentration of 0.3 mM to Scope of these claims and their equivalents be covered induce the expression of target proteins. Mannitol (Sigma, thereby. M1902) was added to each well to a final concentration of 1% US 8,906,636 B2 45 46 to induce the expression of folding modulators in folding TABLE 10-continued modulator over-expressing strains, and the temperature was reduced to 25°C. Twenty four hours after induction, cells Mean CRM197 yield for CRM197-expression strains were normalized to OD600–15 using PBS in a volume of 400 Corresponding Samples were frozen for later processing by Sonication and Strain Number centrifugation to generate soluble and insoluble fractions. in U.S. patent Sample Preparation and SDS-CGE Analysis application Mean Strain Ser. No. Yield Std Dev Soluble and insoluble cellular fractions were prepared by Number 61/325,235 Host Leader (mg/L) (3 replicates) sonication of the normalized cultures followed by centrifu gation. Frozen, normalized culture broth (400 uL) was 10 PS538-779 PS538-749 2 DsbC 567 141 PS538-780 PS538-750 2 ToB 382 217 thawed and sonicated for 3.5 minutes. The lysates were cen PS538-781 PS538-751 3 DsbA 591 230 trifuged at 20,800xg for 20 minutes (4°C.) and the superna PS538-782 PS538-752 3 AZu 1094 543 tants removed using manual or automated liquid handling PS538-783 PS538-753 3 Ibp-S31A 323 143 (soluble fraction). The pellets (insoluble fraction) were fro PS538-784 PS538-754 3 Tpr 419 70 15 PS538-785 PS538-755 3 CupB2 75 74 Zen and then thawed for re-centrifugation at 20,080xg for 20 PS538-786 PS538-756 3 Cup A2 309 214 minutes at 4C, to remove residual Supernatant. The pellets PS538-787 PS538-757 3 NikA 52 73 were then resuspended in 400 uL of 1x phosphate buffered PS538-788 PS538-758 3 Pbp A2OV 356 295 saline (PBS), pH 7.4. Further dilutions of soluble and PS538-789 PS538-759 3 DsbC 319 117 insoluble samples for SDS-CGE analysis were performed in PS538-790 PS538-760 3 ToB 69 88 PS538-791 PS538-761 4 DsbA. 270 106 1x phosphate buffered saline (PBS), pH 7.4. Soluble and PS538-792 PS538-762 4 AZu O 14 insoluble samples were prepared for SDS capillary gel elec PS538-793 PS538-763 4 Ibp-S31A O 6 trophoresis (CGE) (Caliper Life Sciences, Protein Express PS538-794 PS538-764 4. Tpr O O LabChip Kit, Part 760301), in the presence of dithiothreitol PS538-795 PS538-765 4 CupB2 18 39 (DTT). PS538-796 PS538-766 4 Cup A2 118 134 Representative gel-like images showing the results of the PS538-797 PS538-767 4 NikA O 9 25 PS538-798 PS538-768 4 Pbp A2OV O O reducing SDS-CGE analysis of the soluble fraction from each PS538-799 PS538-769 4 DsbC O O strain are shown in FIG. 2. Table 10 shows the mean soluble PS538-800 PS538-770 4 ToIB O O CRM197 yield and standard deviation of 3 replicates for each of the CRM197-expression strains constructed. The host strain and secretion leader screened for each strain are also indicated. 30 Example 2 Both secretion leader and host strain showed a significant impact on CRM197 expression. Expression ranged from no Large-Scale Expression of a Recombinant CRM197 detectable yield to more than 1.2 g/L at the 0.5 mL scale, with Protein the highest expression levels observed in the Host Strain 2 background. The yield observed in PS538-776 was 1263 35 Recombinant CRM197 protein was produced in mg/L, and that in PS538-772 was 1241 mg/L, both well over Pseudomonas fluorescens strains PS538-772, PS538-776, the average yield of 340 mg/L. Both high and low yields were and PS538-782 in 2 liter fermentors. Cultures were grown in observed in the same host straindepending on the leader used, 2 liter fermentors containing a mineral salts medium as and both high and low yields were observed using the same described herein and also by, e.g., Riesenberg, D., et al., 1991, leader in different host strains. 40 and maintained at 32° C. and pH 6.5 through the addition of PS538-772, PS538-773, PS538-776, PS538-778, PS538 ammonia. Dissolved oxygen was maintained in excess 782 were selected for evaluation in large-scale fermentation. through increases in agitation and flow of sparged air and oxygen into the fermentor. Glycerol was delivered to the culture throughout the fermentation to maintain excess levels. TABLE 10 These conditions were maintained until a target culture cell Mean CRM197 yield for CRM197-expression strains 45 density (optical density at 575 nm (A575)) for induction is reached, at which time IPTG is added to initiate CRM197 Corresponding production. Cell density at induction could be varied from Strain Number in U.S. patent A575 of 40 to 200 absorbance units (AU). IPTG concentra application Mean tions could be varied in the range from 0.02 to 0.4 mM. pH Strain Ser. No. Yield Std Dev 50 from 6 to 7.5 and temperature 20 to 35° C. After 16-24 hours, Number 61/325,235 Host Leader (mg/L) (3 replicates) the culture from each bioreactor was harvested by centrifu gation and the cell pellet frozen at -80° C. Samples were PS538-761 PS538-731 1 DsbA. 205 162 PS538-762 PS538-732 1 Azu 427 186 analyzed by SDS-CGE for product formation. PS538-763 PS538-733 1 Ibp-S31A 361 183 Multiple fermentation conditions were evaluated resulting PS538-764 PS538-734 1 Tpr 298 106 55 in top CRM197 expression as determined by SDS-CGE of 1 PS538-765 PS538-735 1 Cup32 105 109 to 2 g/L (see FIGS. 23 and 24). The identities of the induced PS538-766 PS538-736 1 Cup A2 175 99 proteins were confirmed by Western blot analysis using a PS538-767 PS538-737 1 NikA 314 85 PS538-768 PS538-738 1 Pbp A2OV 291 204 diphtheria toxin specific antibody (FIG. 25). PS538-769 PS538-739 1 DsbC 148 91 PS538-770 PS538-740 1 TOIB 213 36 Example 3 PS538-771 PS538-741 2 DsbA. 407 218 60 PS538-772 PS538-742 2 AZu 1241 372 High Throughput Expression of a Recombinant PS538-773 PS538-743 2 Ibp-S31A 1107 219 Cholera Toxin B Protein PS538-774 PS538-744 2 Tpr 28O 285 PS538-775 PS538-745 2 CubE32 192 219 PS538-776 PS538-746 2 Cup A2 1263 474 Construction and Growth of Cholera Toxin B Expression PS538-777 PS538-747 2 NikA 699 259 65 Strains PS538-778 PS538-748 2 Pbp A2OV 914 416 The Cholera Toxin B coding sequence was constructed using Pfluorescens preferred codons to encode the Cholera US 8,906,636 B2 47 48 Toxin Bamino acid sequence. FIG. 3 shows the amino acid TABLE 11-continued and DNA sequences of the expressed synthetic Cholera Toxin B gene. Cholera Toxin B Expression Summary Plasmids carrying the optimized Cholera Toxin B Strain Host Mean Yield Std Dev sequence, fused to the same ten Pfluorescens secretion leader 5 Number Strain Plasmid Leader (mg/L) (3 replicates) coding sequences used with CRM197 (shown in Table 8) were constructed. The secretion leaders were included to PS538-115 4 p538-025 Cup B2 2 4 PSS38-116 4 p538-026 Cup A2 15 16 target the protein to the periplasm for recovery in the properly PS538-117 4 p538-027 NikA O 2 folded and active form. PSS38-118 4 p538-028 Pbp A2OV 35 15 Constructs expressing the ten secretion leaders fused to the 10 PSS38-119 4 p538-029 DsbC O 2 recombinant Cholera Toxin B protein were tested in Pfluo PSS38-120 4 p538-030 TolB O 2 rescens hosts. The four hosts listed in Table 9 were tested with each expression plasmid. Host cells were electroporated with the indicated plasmids, and grown and induced in 96-well Example 4 format as described above for the CRM197 high throughput 15 expression. Samples were prepared and analyzed by SDS Large-Scale Expression of a Recombinant Cholera CGE as described above for the CRM197 high throughput Toxin B Protein expression samples. Representative gel-like images showing the results of the Recombinant Cholera Toxin B protein was produced in reducing SDS-CGE analysis of the soluble fraction from each Pseudomonas fluorescens Pfenex Expression TechnologyTM strain are shown in FIG. 4. Table 11 shows the mean soluble Strains PS538-088 and PS538-091. The Selected Strain was Cholera Toxin B yield and standard deviation of 3 replicates for each of the Cholera Toxin B-expression strains con grown in 2 liter fermentors containing a mineral salts medium structed. as described herein and also by, e.g., Riesenberg, D., et al., Both secretion leader and host strain showed a significant 25 1991, and maintained at 32° C. and pH 6.5 through the addi impact on Cholera Toxin B expression. Expression ranged tion of ammonia. Dissolved oxygen was maintained in excess from no detectable yield to more than 0.2 g/L at the 0.5 mL through increases in agitation and flow of sparged air and scale, with the highest expression levels observed in the oxygen into the fermentor. Glycerol was delivered to the hslUV pre1 degP1 degP2 aprA deletion/DegP2 S219A over culture throughout the fermentation to maintain excess levels. expression host background. Expression of Cholera Toxin B 30 These conditions were maintained until a target culture cell fused to leaders 6 (Cup A2) and 8 (Pbp A20V) appeared to be density (optical density at 575 nm (A575)) for induction was consistently high in all four strains. reached, at which time IPTG was added to initiate the target protein production. IPTG was added to initiate CTB produc TABLE 11 tion. After 16-24 hours, the culture from each bioreactor was 35 harvested by centrifugation and the cell pellet was frozen at Cholera Toxin B Expression Summary -80° C. Strain Host Mean Yield Std Dev Multiple fermentation conditions were evaluated resulting Number Strain Plasmid Leader (mg/L) (3 replicates) in top CTB expression as determined by SDS-CGE of 0.6 to PS538-081 1 p538-021 DsbA 25 8 1.0 g/L. The top performing fermentation cultures were PS538-082 1 p538-022 AZu 1 8 40 induced at approximately 80-160 OD with 0.2 mM IPTG at PS538-083 1 p538-023 Ibp-S31A O 5 PS538-084 1 p538-024. Tpr 35 14 pH 6.5-7.2 and 32° C. Soluble CTB concentrations were PS538-085 1 p538-025 CubE32 10 9 determined by SDS-CGE (see FIG. 14 and Table 12). The PS538-086 1 p538-026 Cup A2 138 18 identities of the induced proteins were confirmed by peptide PS538-087 1 p538-027 NikA O 5 mass fingerprint using MALDI-TOF mass spectrometry. PS538-088 1 538-028 Pbb A2OV 213 23 45 PS538-089 1 538-029 DsbC O 6 PS538-090 1 538-030 TOIB O 4 TABLE 12 PS538-091 2 p538-021 DsbA. 133 62 PS538-092 2 p538-022 AZu 83 56 Soluble Cholera Toxin B Titers PS538-093 2 p538-023 Ibp-S31A 50 44 PS538-094 2 p538-024. Tpr 61 55 50 Product PS538-095 2 538-O2S CB2 62 19 Strain Fermentation Product concentration (gL) PS538-096 2 p538-026 Cup A2 147 57 PS538-097 2 p538-027 NikA 31 28 PS538-088 U5 CTB O.94 O.O3 PS538-098 2 538-028 Pbb A2OV 223 78 PS538-088 U6 CTB O.59 OO1 PS538-099 2 538-029 DsbC 41 24 PS538-091 U3 CTB O.81 - O.O9 PSS38-100 2 538-030 TOIB 6 5 55 PSS38-101 3 p538-021 DsbA. 1 7 PSS38-102 3 p538-022 AZu 1 2 PSS38-103 3 p538-023 Ibp-S31A 19 17 Example 5 PSS38-104 3 p538-024. Tpr 28 36 PS538-105 3 538-O2S CB2 5 9 High Throughput Expression of a Recombinant PSS38-106 3 p538-026 Cup A2 40 12 PS538-107 3 538-027 NikA 5 10 60 Pertussis Toxin Protein PSS38-108 3 538-028 Pbb A2OV 45 19 PSS38-109 3 538-029 DsbC O 6 Construction and Growth of Pertussis Toxoid S1 E129A R9K PSS38-110 3 538-030 TOIB O 3 Expression Strains PSS38-111 4 p538-021 DsbA. O 5 PSS38-112 4 p538-022 AZL O 3 The sequence of the Pertussis toxoid operon encoding Sub PSS38-113 4 p538-023 Ibp-S31A O 2 65 units S1, S2, S3, S4 and S5, with S1 mutations E129A and PSS38-114 4 p538-024. Tpr 13 3 R9K was used for expression of recombinant Pertussis toxin. FIG. 5 shows a map of the operon. FIG. 6 shows the DNA US 8,906,636 B2 49 50 sequence of the operon, with translation (SEQ ID NO:24). strains PS538-321, PS538-324, PS538-325, PS538-326, and FIG. 7 shows the individual amino acid sequences of S1, S2, PS538-328. The selected strain is grown in 2 liter fermentors, S3, S4 and S5. induced with IPTG, and samples prepared for analysis, as The construct was expressed in eight Pfluorescens hosts, described above for CTB large-scale expression. The samples shown in Table 13. Host cells were electroporated with p538 are analyzed by SDS-CGE, for product formation and their 081, and grown and induced in 96-well format as described activity analyzed by Western Blot. above for CRM197 high throughput expression. Samples were prepared and analyzed by SDS-CGE as described above Example 7 for the CRM197 high throughput expression samples. 10 High Throughput Expression of Recombinant TABLE 13 Wild-Type Pertussis Toxoid Pertussis Toxin SI E129A R9K Expression Strains Construction and Growth of Pertussis Toxoid Expression Strains Strain 15 Number Host Genotype Plasmid Type The sequence of the wild-type Pertussis toxin operon PSS38-321 1 lon, la, aprA 538-081 PD encoding subunits S1, S2, S3, S4 and S5, with S1 is used for PSS38-322 2 hslUV, pre1, degP1, 538-081 PD expression of recombinant Pertussis Toxoid. FIG. 17 shows degP2 and aprA the DNA sequence of the operon, with translation (SEQ ID PS538-323 3 disbABCD 538-081 FMO PSS38-324 4 grpE, dinaKJ 538-081 FMO NO:35). PS538-325 5 htpX 538-081 PD The construct is expressed in the Pfluorescens hosts shown PSS38-326 6 RXFO1590 538-081 PD in Table 14. Each strain listed that does not have an overex PS538-327 7 lon, la, aprA deletions; 538-081 PD pression plasmid is tested a) as described (having no overex overexpressesgrpE and FMO pression plasmid); b) including a GrpE DnaKJ overexpres dnaKJ sion plasmid, and c) including a DsbABCD overexpression PSS38-328 8 ppiB (RXFO5345) 538-081 FMO 25 plasmid. Host cells are electroporated with the PTX WT PD = Protease Deletion (listed proteases are deleted); expression plasmid, and grown and induced in 96-well format FMO = Folding Modulator Overexpressor (listed folding modulators are overexpressed. as described above for PTX S1 R9K E129A high-throughput expression. Samples are prepared and analyzed by SDS-CGE Western Blot Analysis of Expressed Pertussis Toxin also as described above. Soluble fractions from the eight cultures described above 30 were analyzed by Western blot to evaluate Pertussis Toxoid expression. Twenty microliters of the soluble fractions (2x TABLE 1.4 diluted, reduced and non-reduced) were run on Bio-Rad 12% Pertussis Toxoid Wild-Type Expression Strains Bis-Tris Gel in 1x Bio Rad MES running buffer. For reduced Western analysis, 1 xXT reducing agent was added. Proteins 35 Host Genotype Type were transferred from SDS-PAGE at 10OV for 60 minutes 9 hsil UV pre2 PD onto a 0.2 um nitrocellulose membrane (Bio Rad, 1620232) 10 hsil UV degP1 PD using 1x NuPAGE Transfer Buffer (Invitrogen, NP0006-1) 11 8. PD with 20% methanol. Membranes were blocked for 1 hour at 12 hslUV pre1 pre2 PD 40 13 on la pro1 pro2 PD room temperature in BlockerTM 1% Casein in PBS (Pierce, 14 RXFO1590 PD 37528). For detection, the diluents were poured off and more 1 on la aprA PD was added containing the combination of 1:1000 dilution 7 on la pre1 degP2 aprA; overexpresses GrpE PD-FMO DnaKJ each of monoclonal antibodies directed against Bordetella 1S RXFO2151 RXFOO428 PD pertussis toxin S4 and S1 (Abcam, catfiab37686 and #37547). 16 on la degP2 PD The blots were incubated with rocking overnight at 4°C. The 45 17 overexpresses DsbAB FMO blots were washed three times with PBS-Tween for 5 minutes 18 overexpresses DsbCD FMO 19 pre1 degP2; overexpresses degP2 S219A PD-FMO each, and were then incubated in more diluent containing a 20 pre1 degP2 clp1 aprA; overexpresses degP2 PD-FMO 1:5,000 dilution of anti-Mouse IgG-Peroxidase derived in S219A goat (Sigma, Cati A4416) at room temperature for 1 hour. The 21 pre1 degP2 lon aprA; overexpresses degP2 PD-FMO blots were washed three times with PBS-Tween (Sigma, 50 S219A 22 pre1 degP2 degP1 aprA; overexpresses degP2 PD-FMO P3563) for 5 minutes each, before color development using S219A Immunopure Metal Enhanced DAB substrate (Pierce, 23 on pre1 degP2 degP1 aprA; overexpresses degP2 PD+ FMO 34065). Multiple subunits were detected by the anti-S1 and S219A anti-S4 antibodies under both reducing and non reducing 2 hslUV pre1 degP2 degP1 aprA; overexpresses PD-FMO 55 degP2 S219A conditions (FIG. 8). The banding pattern of reduced and 25 on la degP2 pre1 aprA; overexpresses degP2 PD-FMO nonreduced samples of the expressed toxoid observed was S219A consistent with that observed for purified Pertussis toxin from 26 degP2; overexpresses SecB PD-FMO strain 165 as reported by Sekura, et al. (J. Biological Chem 27 degP2; overexpresses FkbP PD-FMO 28 degP2; overexpresses GroELES PD-FMO istry 258: 14647, 1983). 29 on la aprA; overexpresses SecB PD-FMO 60 30 on la aprA; overexpresses FkbP PD-FMO Example 6 31 on la aprA; overexpresses GroELES PD-FMO 32 disbC PD Large-Scale Expression of Recombinant Pertussis 33 dsbC; ovrexpresses DsbAB PD-FMO 3 overexpresses DsbABCD FMO Toxin Protein 35 exA aprA PD 65 36 overexpresses Sly D FMO Recombinant Pertussis toxin protein is produced in 37 on hSUV PD Pseudomonas fluorescens Pfenex Expression TechnologyTM US 8,906,636 B2 51 52 TABLE 14-continued Constructs expressing the ten secretion leaders fused to the recombinant Tetanus Toxin C protein were tested in Pfluo Pertussis TOXoid Wild-Type Expression Strains rescens hosts. The four hosts listed in Table 9 were tested with Host Genotype Type each leader. Host cells were electroporated with the indicated plasmids, and grown and induced in 96-well format as 38 Wt 39 aprA PD described above for the CRM197 high throughput expres 4 overexpresses GrpE DnaKJ FMO sion. Samples were prepared and analyzed by SDS-CGE as 5 htpX PD described above for the CRM197 high throughput expression 40 lon PD samples. 41 pre1 PD 10 42 hSUV PD Representative gel-like images showing the results of the 43 degP2 PD 44 degP1 PD reducing SDS-CGE analysis of the soluble fraction from each 45 pre2 PD strain are shown in FIG. 10. Table 15 shows the mean soluble 46 RXF64S1 PD Tetanus Toxin C yield and standard deviation of 3 replicates 6 RXF1590 PD 15 48 RXF4692 PD for each of the Tetanus Toxin C-expression strains con 49 hSUV mic PD structed. Tetanus Toxin C fragment appeared to be expressed SO RXF2161 PD well in most strains tested, with highest yields ranging up to S1 RXFOO133 PD 600 mg/L in the hslUV pre 1 degP1 degP2 aprA deletion/ 52 RXF2796 PD 53 RXF4968 PD DegP2 S219A overexpression expression host. Strains 54 overexpresses DsbC FMO PS538-529, PS538-538, PS538-544, PS538-546, PS538 55 overexpresses DsbAC FMO 547, PS538-548, PS538-558, PS538-565 and PS538-568 56 overexpresses LepB FMO 57 overexpresses SecB FMO were selected for further evaluation. 58 overexpresses Clp A FMO 59 overexpresses FkB2 FMO TABLE 1.5 60 overexpresses DnaK-like FMO 25 61 overexpresses FkbP FMO 62 overexpresses PpiA FMO Tetanus Toxin C Expression Summary. 8 overexpresses PpiB FMO Strain Mean Yield Std Dev 63 overexpresses HscA FMO 64 overexpresses Gish.A FMO Number Host Plasmid Leader (mg/L) (3 replicates) 65 overexpresses Gor FMO 30 PS538-529 1 p538-132 DsbA 261 75 66 overexpresses TrxC FMO PS538-530 1 p538-133 Azu 200 82 67 overexpresses DsbG FMO 68 overexpresses Ppi FMO PSS38-531 1 p538-134 Ibp-S31A 16S 64 69 overexpresses GroELES FMO PS538-532 1 p538-135 Tpr 2O7 107 70 pre1 aprA; overexpresses GrpE DnaKJ PD-FMO PS538-533 1 p538-136 CubE32 205 128 PSS38-534 1 p538-137 Cup A2 200 117 71 hypersecretion 35 72 overexpresses DsbD FMO PS538-535 1 p538-138 NikA 174 96 73 hypersecretion PS538-536 1 538-139 Pbb A2OV 311 156 74 hypersecretion PS538-537 1 p538-140 DsbC 188 97 75 pre1 pre2 PD PS538-538 1 p538-141 ToB 129 63 76 hslUV clpA PD PS538-539 2 p538-132 DsbA. 486 89 PSS38-540 2 p538-133 Azu 495 93 *Each strain listed that does not have an overexpression plasmid is tested a) as described 40 (having no overexpression plasmid); b) including a GrpEDnaKJ overexpression plasmid, PSS38-541 2 p538-134 Ibp-S31A 568 68 and c) including a DsbABCD overexpression plasmid, PD = Protease Deletion (listed proteases are deleted); PSS38-542 2 p538-135 Tpr 589 364 FMO = Folding Modulator Overexpressor (listed folding modulators are overexpressed. PSS38-543 2 p538-136 CubE32 534 3.18 PSS38-544 2 p538-137 Cup A2 SO4 134 Hypersecretion strains, also known as hyper-vesiculating PS538-545 2 p538-138 NikA 444 145 strains, are described, e.g., in WO2010/008764, "Pseudomo 45 PSS38-546 2 538-139 Pbb A2OV 637 28O nas Fluorescens Strains for Production of Extracellular PS538-547 2 p538-140 DsbC 574 68 Recombinant Protein, incorporated herein by reference in its PSS38-548 2 p538-141 ToB 438 61 entirety. PSS38-549 3 p538-132 DsbA. 174 37 PS538-550 3 p538-133 Azu 18O 58 Example 8 50 PS538-551 3 p538-134 Ibp-S31A 88 58 PS538-552 3 p538-135 Tpr 247 134 PS538-553 3 p538-136 CubE32 199 39 High Throughput Expression of a Recombinant PS538-554 3 p538-137 Cup A2 16S 69 Tetanus Toxin Fragment C Protein PS538-555 3 p538-138 NikA 97 90 PS538-556 3 538-139 Pbb A2OV 297 112 Construction and Growth of Tetanus Toxin C Expression 55 PS538-557 3 p538-140 DsbC 151 52 Strains PS538-558 3 p538-141 ToB 35 13 The Tetanus Toxin C coding sequence was constructed PS538-559 4 p538-132 DsbA. 39 39 using Pfluorescens preferred codons to encode the Tetanus PS538-560 4 p538-133 Azu 40 43 Toxin Camino acid sequence. FIG. 9 shows the amino acid PS538-561 4 p538-134 Ibp-S31A 36 40 60 PS538-562 4 p538-135 Tpr 35 39 and DNA sequences of the expressed synthetic Tetanus Toxin PS538-563 4 p538-136 Cup.32 S4 26 C gene. PSS38-564 4 p538-137 Cup A2 42 36 Plasmids carrying the optimized Tetanus Toxin C PS538-565 4 p538-138 NikA 44 37 sequence, fused to the same ten Pfluorescens secretion leader PSS38-566 4 b538-139 Pbb A2OV 37 40 coding sequences used with CRM197 (shown in Table 8) PS538-567 4 p538-140 DsbC 39 43 were constructed. The secretion leaders were included to 65 PS538-568 4 p538-141 ToB 45 38 target the protein to the periplasm for recovery in the properly folded and active form. US 8,906,636 B2 53 54 Example 9 by SDS-CGE as described above for the CRM197 high throughput expression samples. Large-Scale Expression of a Recombinant Tetanus Toxin Fragment C Protein TABLE 17 Recombinant Tetanus Toxin C protein was produced in TcdB Host Strains Pseudomonas fluorescens Pfenex Expression TechnologyTM strains PS538-529, PS538-538, PS538-544, PS538-546, Host Strain Genotype Type PS538-547, PS538-548, PS538-558, PS538-565 and PS538 37 his UV lon PD 38 T 568. The selected strains were grown in 2 liter fermentors 10 containing a mineral salts medium as described above for 4 dnaKJ grpE FMO CRM197. 5 htpX PD 40 Oil PD Multiple fermentation conditions were evaluated resulting 41 C PD in top soluble TTC expression from strains PS538-529, 42 hSUV PD PS538-546, and PS538-547 of 6 to 10 g/L as determined by 43 degP2 PD SDS-CGE (see FIG. 15 and Table 16). The top performing 15 44 degP1 PD fermentation culture was induced at approximately 160 OD 45 bro2 PD 47 RXFO1590 PD with 0.2 mM IPTGat pH 7.2 and 32°C. The identities of the 49 hsOV mic PD induced proteins were confirmed by peptide mass fingerprint 53 RXFO4968 PD using MALDI-TOF mass spectrometry and Western Blot. 55 disbAC FMO Mass spectrometry and Western blot analyses indicated that 61 kbP FMO the secretion leaders of PS538-529, PS538-546 and PS538 66 ErxC FMO 72 disbD FMO 547 (DsbA, Pbp A20V and DsbC, respectively) were not 76 hslUV clip A PD processed from 100% of the expressed protein under these 12 hslUV pre1 pre2 PD expression conditions. However, the TolB leader was identi 1 on la aprA PD fied as being precisely cleaved from the secreted protein (data 25 16 on la degP2 PD not shown). TolB-TTC expression strains PS538-538, 2 hslUV pre1 degP1 degP2 aprA PD PS538-548, PS538-558 and PS538-568 were screened at the deletions; overexpresses degP2 FMO 2 L fermentation scale, using the conditions outlined above, S219A to identify a strain that enabled production of TTC with high 3 disbABCD FMO fidelity cleavage of the secretion leader and low levels of 21 on pre1 degP2 aprA deletions with PD degradation. Strains PS538-538, PS538-548 and PS538-558 30 degP2S219A overexpression FMO were observed to produce similar quality and yield of material by Western blot analysis (FIG. 15B). Representative gel-like images showing the results of the TABLE 16 reducing SDS-CGE analysis of the soluble fraction from each 35 of the 24 strains tested are shown in FIG. 12. Table 18 shows Soluble Tetanus Toxin C (TTC) Titers the mean soluble TcdB yield and standard deviation of 3 Product replicates for each of the TcdB-expression strains con concentration structed. Strains PS538-654, PS538-659, PS538-669, PS538 Strain Fermentation Product (gL) 671, and PS538-674 were selected for further evaluation. 40 PS538-529 U1 - FIG. 1SA TTC 5.7 1.3 PSS38-546 U7 - FIG. 1SA TTC 9.51.1 TABLE 1.8 PS538-547 US - FIG. 1SA TTC 6.21.9 PS538-538 U1 - FIG. 1SB TTC 2.5 + 0.09 TcdB Expression Summary PS538-538 U2 - FIG. 1SB TTC 18 O2 Strain Mean Yield Std Dew PSS38-548 U3 - FIG. 1SB TTC 5.3 0.6 45 PSS38-548 U4 - FIG. 1SB TTC 4.5 O2 Number Host Plasmi (mg/L) (3 replicates) PS538-558 US - FIG. 1SB TTC 1.1 - 0.8 PS538-558 U6 - FIG. 1SB TTC 1.90.1 PS538-651 37 b538-2 103 7 PSS38-568 U7 - FIG. 1SB TTC O2 OO1 PS538-652 38 S38-2 55 4 PSS38-568 U8 - FIG. 1SB TTC O2 OO1 PS538-653 4 p538-2 57 1 PS538-654 S 538-2 166 13 50 PS538-655 40 p538-2 88 3 PS538-656 41 p538-2 68 5 PS538-657 42 p538-2 90 14 Example 10 PS538-658 43 p538-2 68 2 PS538-659 44 p538-2 109 8 High Throughput Expression of a Recombinant C. PS538-660 45 538-2 78 4 55 PS538-661 6 p538-2 98 15 difficile B Protein PS538-662 49 p538-2 106 10 PS538-663 S3 S38-2 91 6 Construction and Growth of TcdB Expression Strains PS538-664 SS S38-2 45 4 The TcdB coding sequence was constructed using Pfluo PS538-665 61 p538-2 63 6 PS538-666 66 p538-2 56 8 rescens preferred codons to encode the TcdB amino acid PS538-667 72 S38-2 70 8 sequence. FIG. 11 shows the amino acid and DNA sequences 60 PS538-668 76 b538-2 8O 6 of the expressed synthetic TcdB gene. PS538-669 12 538-2 117 39 Plasmids carrying the optimized TcdB sequence were PS538-670 1 p538-2 108 18 PS538-671 16 p538-2 247 65 tested in the Pfluorescens hosts having genotypes listed in PS538-672 2 538-2 32 6 Table 17. Host cells were electroporated with the cytoplasmic PS538-673 3 S38-2 52 2 expression plasmid p538-211, and grown and induced in 65 PS538-674 21 S38-2 145 12 96-well format as described above for the CRM197 high throughput expression. Samples were prepared and analyzed US 8,906,636 B2 55 56 Example 11 TABLE 20-continued Large-Scale Expression of Recombinant C. difficile Exotoxin A Host Strains Toxin B Protein Host Strain Genotype Type Recombinant C. difficile toxin B protein was produced in 8 ppiB PD 1 Lon-, La-, aprA- PD Pseudomonas fluorescens Pfenex Expression TechnologyTM 2 hslUV-, pre1-, degP1-, degP2-, PD strain PS538-654, PS538-659, PS538-669, PS538-671, and aprA- FMO PS538-674. The selected strains were grown in 2 liter fermen with degP2S219A overexpression tors, induced with IPTG, and samples prepared for analysis, 10 3 dsbABCD overexpression FMO as described above for CTB large-scale expression. 4 grpE, dinaKJ overexpression FMO Multiple fermentation conditions were evaluated resulting in top C. difficile B Toxin expression as determined by SDS CGE of approximately 2 g/L. The top performing fermenta Host cells were electroporated with the indicated plasmids, tion culture was induced at approximately 160 OD with 0.08 15 and grown and induced in 96-well format as described above mM IPTG at pH 6.5 and 32° C. Soluble C. difficile B Toxin for the CRM197 high throughput expression. Samples were concentrations were determined by SDS-CGE (see FIG. 16 prepared and analyzed by SDS-CGE as described above for and Table 19). The identities of the induced proteins were the CRM197 high throughput expression samples. The high confirmed by Western blot. est yields ranged from 1.6 to 2.2 g/L of soluble Exotoxin A protein. Table 21 shows the soluble rEPA yield for each of the TABLE 19 expression Strains selected for further testing.
Soluble C. dificile B Toxin (TedB) Titers TABLE 21 Product rEPA HTP Expression Summary Strain Fermentation Product concentration (gL) 25 PS538-671 U5 TcdB 1.6-0.4 Strain Secretion Volumetric PS538-671 U6 TcdB 2.1 O2 Number Host Plasmid Leader Yield (g/L) PS538-674 U7 TcdB 18 O2 PS538-1670 3 S38-2SO DsbC 6.7 PSS38-1663 3 S38-243 Ibp-s31a 5.7 30 PSS38-1633 1 S38-243 Ibp-s31a 5.7 PSS38-1640 1 b538-249 Pb-A2OV 4.7 Example 12 PSS38-1662 3 b538-242 AZu 4.2 PSS38-1632 1 b538-242 AZu 3.2 High Throughput Expression of a Recombinant PS538-1671 4 b538-241 DsbA. 2.9 Exotoxin A Protein PSS38-1665 3 S38-245 Tpr 2.7 35 PSS38-1667 3 S38-247 Cup.A2 2.6 PSS38-1674 4 b538-244 ToB 2.3 Construction and Growth of P aeruginosa Exotoxin A PS538-1672 4 b538-242 AZu 2.2 Expression Strains PSS38-1676 4 b538-246 Cup.32 2.2 The P. aeruginosa Exotoxin A mutant rEPA coding PS538-1677 4 S38-247 Cup.A2 2.1 sequence was constructed using P. fluorescens preferred PS538-1635 1 S38-245 Tpr 2.O codons to encode the rEPA amino acid sequence. FIG. 13 PS538-1675 4 S38-245 Tpr 2.O 40 PS538-1673 4 S38-243 Ibp-s31a 2.O shows the amino acid and DNA sequences of the expressed PSS38-1680 4 S38-2SO DsbC 1.9 synthetic rEPA gene. PS538-1679 4 b538-249 Pb-A2OV 1.7 Plasmids carrying the optimized sequences encoding PSS38-1669 3 b538-249 Pb-A2OV 1.6 either the deletion mutant rEPA, as indicated in FIG. 13, fused PSS38-1678 4 b538-248 NikA 1.5 to the same ten P. fluorescens secretion leader coding PSS38-1652 2 b538-242 AZu 1.5 45 PS538-1653 2 S38-243 Ibp-s31a 1.4 sequences used with CRM197 (shown in Table 8) were con PSS38-1660 2 S38-2SO DsbC 1.4 structed. The Secretion leader coding sequences were PS538-1637 1 S38-247 Cup.A2 1.3 included to target the protein to the periplasm for recovery in PSS38-1666 3 b538-246 Cup.32 1.1 the properly folded and active form. PSS38-1636 1 b538-246 Cup.32 1.O Constructs expressing the ten secretion leaders fused to the PSS38-1634 1 b538-244 ToB 1.O rEPA proteins were tested in eight Pfluorescens hosts, listed 50 PS538-1627 8 S38-247 Cup.A2 O.8 PSS38-1631 1 b538-241 DsbA. O.8 in Table 20. Host cells were electroporated with the indicated PSS38-1622 8 b538-242 AZu O.8 plasmids, and grown and induced in 96-well format as PSS38-1661 3 b538-241 DsbA. 0.7 described above for the CRM197 high throughput expres PSS38-1603 5 S38-243 Ibp-s31a O.6 sion. Samples were prepared and analyzed by SDS-CGE as PSS38-1630 8 S38-2SO DsbC O.6 described above for the CRM197 high throughput expression 55 PSS38-16O2 5 b538-242 AZu O.6 PSS38-1605 5 S38-245 Tpr O.6 samples. The highest yields ranged from 4.7-6.7 g/L of PSS38-1623 8 S38-243 Ibp-s31a O.6 Soluble rEPA. PSS38-1664 3 b538-244 ToB O.S PSS38-1668 3 b538-248 NikA O.S TABLE 20 PSS38-1610 5 S38-2SO DsbC O.S PSS38-1606 5 b538-246 Cup.32 0.4 60 Exotoxin A Host Strains PSS38-1659 2 b538-249 Pb-A2OV 0.4 PS538-1607 5 S38-247 Cup.A2 0.4 Host Strain Genotype Type PSS38-1626 8 b538-246 Cup.32 0.4 PSS38-1625 8 S38-245 Tpr 0.4 5 htpX PD PSS38-1638 1 b538-248 NikA O.3 6 Serralysin PD PSS38-1609 5 b538-249 Pb-A2OV O.3 7 Lon La1 aprA; with grpEdnaKJ PDFMO 65 PSS38-1604 5 b538-244 ToB O.3 overexpress ion PSS38-1629 8 b538-249 Pb-A2OV O.3 US 8,906,636 B2 57 58 TABLE 21-continued TABLE 22 rEPA HTP Expression Summary rEPA Fermentation Analysis Strain Secretion Volumetric Strain Number Fermentation Yield (g/L) Number Host Plasmid Leader Yield (g/L) PSS38-1633 U1 15.5 +f- 0.7 PS538-1657 2 S38-247 Cup.A2 O.2 PSS38-1633 U2 11.1 +/- 0.6 PSS38-1651 2 b538-241 DsbA. O.2 PSS38-1640 U3 20.1 +/- 1.7 PSS38-1601 5 b538-241 DsbA. O.2 PSS38-1640 U5 31.9 +/- 1.6 PSS38-1624 8 b538-244 ToB O.2 PS538-1670 U6 20.0 +/- 0.7 PSS38-1621 5 b538-241 DsbA. O.2 10 PS538-1670 U7 14.6 +/- 1.1 PSS38-1608 5 b538-248 NikA O.2 PS538-1670 U8 31.0 +/- 1.7 PSS38-1654 2 b538-244 ToB O.2 PSS38-1628 8 b538-248 NikA O.1 PSS38-1658 2 b538-248 NikA O.1 PS538-1655 2 S38-245 Tpr O.1 Example 14 PSS38-1641 7 b538-241 DsbA. O.1 15 PSS38-1611 6 b538-241 DsbA. NQ PSS38-1612 6 b538-242 AZu NQ High Throughput Expression of a Recombinant PSS38-1613 6 S38-243 Ibp-s31a NQ Wild-Type Diphtheria Toxin Protein PSS38-1614 6 b538-244 ToB NQ PSS38-1615 6 S38-245 Tpr NQ PSS38-1616 6 b538-246 Cup.32 NQ Construction and Growth of Wild-Type Diphtheria Toxin PS538-1617 6 S38-247 Cup.A2 NQ Expression Strains PSS38-1618 6 b538-248 NikA NQ PSS38-1619 6 b538-249 Pb-A2OV NQ A Diphtheria Toxin coding sequence is constructed using PSS38-1620 6 S38-2SO DsbC NQ Pfluorescens preferred codons to encode the wild-type Diph PSS38-1642 7 b538-242 AZu NQ theria Toxin amino acid sequence. FIG. 18 shows the amino PSS38-1643 7 S38-243 Ibp-s31a NQ acid and DNA sequences of the expressed synthetic Diphthe PSS38-1644 7 b538-244 ToB NQ 25 PSS38-1645 7 S38-245 Tpr NQ ria Toxin gene. PSS38-1646 7 b538-246 Cup.32 NQ Plasmids carrying the optimized sequences encoding PSS38-1647 7 S38-247 Cup.A2 NQ PSS38-1648 7 b538-248 NikA NQ Diphtheria Toxin, fused to the ten Pfluorescens secretion PSS38-1649 7 b538-249 Pb-A2OV NQ leader coding sequences used with CRM197 (shown in Table PSS38-16SO 7 S38-2SO DsbC NQ 30 8) are constructed. The secretion leader coding sequences are PSS38-1656 2 b538-246 Cup.32 NQ included to target the protein to the periplasm for recovery in NQ = not quantifiable the properly folded and active form. Constructs expressing the ten secretion leaders fused to the recombinant Diphtheria Toxin proteins are tested in Pfluo Example 13 35 rescens hosts. The four hosts listed in Table 9 are tested with each leader. Host cells are electroporated with the indicated plasmids, and grown and induced in 96-well format as Large-Scale Expression of a Recombinant described above for the CRM197 high throughput expres Pseudomonas aeruginosa Exotoxin A Protein sion. Samples are prepared and analyzed by SDS-CGE as 40 described above for the CRM197 high throughput expression Recombinant Paeruginosa exotoxin A protein (rEPA) was samples. produced in Pseudomonas fluorescens strains PS538-1633, Example 15 PS538-1640 and PS538-1670 in 2 liter fermentors. Cultures were grown in 2 liter fermentors containing a mineral salts 45 Large-Scale Expression of a Recombinant medium as described herein and also by, e.g., Riesenberg, D., Wild-Type Diphtheria Toxin Protein et al., 1991, and maintained at 32° C. and pH 6.5 through the addition of ammonia. Dissolved oxygen was maintained in Recombinant Wild-Type Diphtheria Toxin protein is pro excess through increases in agitation and flow of sparged air duced in selected Pseudomonas fluorescens Pfenex Expres and oxygen into the fermentor. Glycerol is delivered to the 50 sion TechnologyTM strains. The selected strains are grown in culture throughout the fermentation to maintain excess levels. 2 liter fermentors, induced with IPTG, and samples prepared These conditions were maintained until a target culture cell for analysis, as described above for CRM197 large-scale density (optical density at 575 nm (A575)) for induction is expression. The samples are analyzed by SDS-CGE. reached, at which time IPTG was added to initiate rEPApro duction. Cell density at induction can be varied from A575 of 55 Example 16 40 to 200 absorbance units (AU). IPTG concentrations can be varied in the range from 0.02 to 0.4 mM. pH from 6 to 7.5 and High Throughput Expression of a Recombinant temperature 20 to 35°C. After 16-24 hours, the culture from Cholera Holotoxin Protein each bioreactor was harvested by centrifugation and the cell pellet frozen at -80°C. Samples were analyzed by SDS-CGE 60 Construction and Growth of CTX Expression Strains for product formation. The CTX coding sequence is constructed using Pfluore Multiple fermentation conditions were evaluated resulting scens preferred codons to encode the CTX amino acid in top rEPA expression as determined by SDS-CGE of up to sequence. The coding sequence is based on the amino acid 32 g/L (FIGS. 20 and 21). The identity of the induced protein and DNA sequences of the CTX gene shown in FIG. 19. was confirmed by Western blot analysis using an antibody 65 Plasmids carrying the optimized CTX sequence, fused to specific for P. aeruginosa exotoxin A (FIG. 22). The yields the ten Pfluorescens Secretion leader coding sequences used obtained are shown in Table 22. with CRM197 (shown in Table 8) are constructed. The secre US 8,906,636 B2 59 60 tion leaders are included to target the protein to the periplasm TABLE 23-continued for recovery in the properly folded and active form. Constructs expressing the ten secretion leaders fused to the Sequence Listings recombinant CTX protein are tested in Pfluorescens hosts. The four hosts listed in Table 9 are tested with each expression 5 SEQID NO DESCRIPTION plasmid. Host cells are electroporated with the indicated plas- 11 DsbC mids, and grown and induced in 96-well format as described . above for the CRM197 high throughput expression. Samples 14 re are prepared and analyzed by SDS-CGE as described above 15 CupC2 for the CRM197 high throughput expression samples. 10 16 Pore 17 Pbp 18 Flg.I Example 17 19 ttg2C 2O CRM197 native leader Large-Scale Expression of a Recombinant Cholera 21 Cleavage product GADD Holotoxin Protein 15 22 Cholera Toxin BAmino Acid Sequence 23 Cholera Toxin B DNA Sequence, optimized 24 Pertussis toxin S1 R9K E129A DNA sequence Recombinant Cholera Holotoxin protein is produced 1. 25 Pertussis toxin S1 R9K E129A Amino Acid Sequence selected Pseudomonas fluorescens Pfenex Expression Tech- 26 Pertussis toxin S2 Amino Acid Sequence nologyTM strains. The selected strains are grown in 2 liter 27 Pertussis toxin S3 Amino Acid Sequence fermentors, induced with IPTG, and samples prepared for 20 28 Pertussis toxin S4 Amino Acid sequence analysis, as described above for CRM197 large-scale expres- 29 Pertussis toxin SS Amino Acid sequence sion. The samples are analyzed by SDS-CGE 30 Tetanus Toxin CAmino Acid Sequence p y y 31 Tetanus Toxin C DNA Sequence, optimized 32 TcdB Amino Acid Sequence TABLE 23 33 TcdB DNA Sequence,C optimizedp 25 34 Exotoxin A Amino Acid Sequence Sequence Listings 35 DNA Sequence of Wild-Type Pertussis Toxoid 36 Wild-Type Diphtheria Toxin Amino Acid Sequence SEQID NO DESCRIPTION 37 Wild-Type Diphtheria Toxin DNA Sequence, optimized 1 CRM197 Amino Acid Sequence 38 Cholera Toxin A Amino Acid Sequence 39 Cholera Toxin BAmino Acid Sequence 2 CRM197 DNA Sequence, optimized 40 Cholera Holotoxin (CTX) DNAS 3 DsbA Secretion Leader 30 tolera Holotoxin ( ) sequence 4 AZu 41 Wild Type Pertussis toxin S1 Amino Acid Sequence 5 Ibp-S31A 42 Pertussis toxin S2Amino Acid Sequence 6 Tpr 43 Pertussis toxin S4 Amino Acid Sequence 7 CupB2 44 Pertussis toxin S5 Amino Acid Sequence 8 Cup A2 45 Pertussis toxin S3 Amino Acid Sequence 9 NikA 35 46 Hexa-histidine affinity tag 10 Pbp A2OV
SEQUENCE LISTING
<16 Os NUMBER OF SEO ID NOS: 46
<21 Os SEQ ID NO 1 &211s LENGTH: 535 212s. TYPE: PRT <213> ORGANISM: Corynebacterium diphtheriae
<4 OOs SEQUENCE: 1 Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Wal Met Glu Asn 1. 5 1O 15 Phe Ser Ser Tyr His Gly. Thir Lys Pro Gly Tyr Val Asp Ser Ile Glin 2O 25 3 O Lys Gly Ile Glin Llys Pro Llys Ser Gly Thr Glin Gly Asn Tyr Asp Asp 35 4 O 45 Asp Trp Llys Glu Phe Tyr Ser Thr Asp Asn Llys Tyr Asp Ala Ala Gly SO 55 60
Tyr Ser Val Asp Asn. Glu ASn Pro Lieu. Ser Gly Lys Ala Gly Gly Val 65 70 7s 8O
Val Llys Val Thr Tyr Pro Gly Lieu. Thir Lys Val Lieu Ala Lieu Lys Val 85 90 95
Asp Asn Ala Glu Thir Ile Llys Lys Glu Lieu. Gly Lieu. Ser Lieu. Thr Glu 1OO 105 110 US 8,906,636 B2 61 62 - Continued
Pro Luell Met Glu Glin Wall Gly Thir Glu Glu Phe Ile Lys Arg Phe Gly 115 12 O 125
Asp Gly Ala Ser Arg Wall Wall Luell Ser Luell Pro Phe Ala Glu Gly Ser 13 O 135 14 O
Ser Ser Wall Glu Tyr Ile Asn Asn Trp Glu Glin Ala Lys Ala Luell Ser 145 150 155 160
Wall Glu Luell Glu Ile Asn Phe Glu Thir Arg Gly Arg Gly Glin Asp 1.65 17s
Ala Met Glu Tyr Met Ala Glin Ala Ala Gly Asn Arg Wall Arg 18O 185 19 O
Arg Ser Wall Gly Ser Ser Lell Ser Ile ASn Lell Asp Trp Asp Wall 195
Ile Arg Asp Thir Thir Ile Glu Ser Lell Glu His Gly 21 O 215
Pro Ile Asn Met Ser Glu Ser Pro ASn Thir Wall Ser Glu 225 23 O 235 24 O
Glu Ala Glin Tyr Lell Glu Glu Phe His Glin Thir Ala Luell Glu 245 250 255
His Pro Glu Luell Ser Glu Lell Thir Wall Thir Gly Thir Asn Pro Wall 26 O 265 27 O
Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Wall Asn Wall Ala Glin Wall 285
Ile Asp Ser Glu Thir Ala Asp Asn Luell Glu Lys Thir Thir Ala Ala Luell 29 O 295 3 OO
Ser Ile Luell Pro Gly Ile Gly Ser Wall Met Gly Ile Ala Asp Gly Ala 3. OS 310 315
Wall His His Asn Thir Glu Glu Ile Wall Ala Glin Ser Ile Ala Luell Ser 3.25 330 335
Ser Luell Met Wall Ala Glin Ala Ile Pro Luell Wall Gly Glu Luell Wall Asp 34 O 345 35. O
Ile Gly Phe Ala Ala Asn Phe Wall Glu Ser Ile Ile Asn Luell Phe 355 360 365
Glin Wall Wall His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly His 37 O 375
Lys Thir Glin Pro Phe Lell His Asp Gly Tyr Ala Wall Ser Trp Asn Thir 385 390 395 4 OO
Wall Glu Asp Ser Ile Ile Arg Thir Gly Phe Glin Gly Glu Ser Gly His 4 OS 41O 415
Asp Ile Ile Thir Ala Glu Asn Thir Pro Luell Pro Ile Ala Gly Wall 425 43 O
Lell Luell Pro Thir Ile Pro Gly Lys Luell Asp Wall Asn Lys Ser Thir 435 44 O 445
His Ile Ser Wall Asn Gly Arg Ile Arg Met Arg Arg Ala Ile 450 45.5 460
Asp Gly Asp Wall Thir Phe Arg Pro Ser Pro Wall Wall Gly 465 470 48O
Asn Gly Wall His Ala Asn Lell His Wall Ala Phe His Arg Ser Ser Ser 485 490 495
Glu Ile His Ser Asn Glu Ile Ser Ser Asp Ser Ile Gly Wall Luell SOO 505 51O US 8,906,636 B2 63 - Continued Gly Tyr Glin Llys Thr Val Asp His Thr Llys Val Asn. Ser Lys Lieu. Ser 515 525 Lieu. Phe Phe Glu Ile Llys Ser 53 O 535
SEQ ID NO 2 LENGTH: 1605 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic polynucleotide FEATURE: NAME/KEY: CDS LOCATION: (1) ... (1605)
<4 OOs, SEQUENCE: 2
999 gcg gac gat gtg gtg gat to c to c aag tog titt gtc atg gala aat 48 Gly Ala Asp Asp Wall Wall Asp Ser Ser Lys Ser Phe Wall Met Glu Asn 1. 5 1O 15 tto tog tog tac Cat ggc act aag CC a ggc tac gtg gat agc att Cala 96 Phe Ser Ser Tyr His Gly Thir Lys Pro Gly Wall Asp Ser Ile Glin 2O 25 3O aag ggc at C cag aag c cc aag agc ggt act cag 999 aac tat gac gac 144 Lys Gly Ile Glin Lys Pro Lys Ser Gly Thir Glin Gly Asn Asp Asp 35 4 O 45 gac tgg aag gala titt tac agc acc gac aat aag tac gat gct gcg ggc 192 Asp Trp Lys Glu Phe Ser Thir Asp Asn Lys Tyr Asp Ala Ala Gly SO 55 6 O tat agc gtg gac c gaa c CC ttg tcg 93C aag gCC ggit 93C gtg 24 O Tyr Ser Wall Asp Asn Glu Asn Pro Luell Ser Gly Lys Ala Gly Gly Wall 65 70 8O gtg aag gtg acc tat cott ggit Ctg acg a.a.a. gtt Ctg gcg ttg a.a.a. gtg 288 Wall Lys Wall Thir Tyr Pro Gly Luell Thir Lys Wall Lell Ala Luell Lys Wall 85 90 95 gac aac gcc gag act atc. aag a.a.a. gala ggc ttg agt ttg acc gag 336 Asp Asn Ala Glu Thir Ile Lys Glu Luell Gly Lell Ser Luell Thir Glu 1OO 105 11 O cc.g Ctg atg gala Cag gtg ggit acc gala gala titc att a.a.a. cgt. titt 999 384 Pro Luell Met Glu Glin Wall Gly Thir Glu Glu Phe Ile Lys Arg Phe Gly 115 12 O 125 gac ggc gcg tog cgc gtg gtc Ctg tog ttg cc.g tto gcc gala 999 tog 432 Asp Gly Ala Ser Arg Wall Wall Luell Ser Luell Pro Phe Ala Glu Gly Ser 13 O 135 14 O tcg tog gtg gala tat atc. aac aac tgg gala cag gcc aag gcg Ctg tog Ser Ser Wall Glu Tyr Ile Asn Asn Trp Glu Glin Ala Ala Luell Ser 145 150 155 160 gtg gala Ctg gala att aac tto gala acg cgg ggc a.a.a. cgg ggc cag gac 528 Wall Glu Luell Glu Ile Asn Phe Glu Thir Arg Gly Arg Gly Glin Asp 1.65 17O 17s gcc atg tac gala tac atg gcg cag gcg gcc 999 aac cgg gtg cgg 576 Ala Met Glu Tyr Met Ala Glin Ala Ala Gly Asn Arg Wall Arg 18O 185 19 O cgc agc gtg ggc agt t cc ttg tgc at C aat Ctg gac tgg gac gt C 624 Arg Ser Wall Gly Ser Ser Lell Ser Ile ASn Lell Asp Trp Asp Wall 195 2OO 2O5 atc. cgc gat aag acg aag acg a.a.a. at C gag tog citc. a.a.a. gag CaC ggc 672 Ile Arg Asp Thir Thir Ile Glu Ser Lell Glu His Gly 21 O 215 22O cc.g at C a.a.a. aac a.a.a. atg agc gag tog cc.g aat a.a.a. acg gtg to c gag 72 O Pro Ile Asn Lys Met Ser Glu Ser Pro ASn Thir Wall Ser Glu 225 23 O 235 24 O
US 8,906,636 B2 67 68 - Continued
<210s, SEQ ID NO 3 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide
<4 OOs, SEQUENCE: 3 Met Arg Asn Lieu. Ile Lieu. Ser Ala Ala Lieu Val Thr Ala Ser Lieu. Phe 1. 5 1O 15
Gly Met Thr Ala Glin Ala 2O
<210s, SEQ ID NO 4 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide
<4 OOs, SEQUENCE: 4 Met Phe Ala Lys Lieu Val Ala Val Ser Lieu. Lieu. Thir Lieu Ala Ser Gly 1. 5 1O 15
Gln Lieu. Lieu. Ala 2O
<21Os SEO ID NO 5 &211s LENGTH: 31 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide
<4 OOs, SEQUENCE: 5 Met Ile Arg Asp Asn Arg Lieu Lys Thir Ser Lieu. Lieu. Arg Gly Lieu. Thir 1. 5 1O 15
Lieu. Thir Lieu. Lieu. Ser Lieu. Thir Lieu. Lieu Ser Pro Ala Ala His Ala 2O 25 3O
<210s, SEQ ID NO 6 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide
<4 OOs, SEQUENCE: 6 Met Asn Arg Ser Ser Ala Lieu. Lieu. Lieu Ala Phe Val Phe Lieu. Ser Gly 1. 5 1O 15
Cys Glin Ala Met Ala 2O
<210s, SEQ ID NO 7 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide US 8,906,636 B2 69 70 - Continued
<4 OO > SEQUENCE: 7 Met Lieu. Phe Arg Thr Lieu. Lieu Ala Ser Lieu. Thir Phe Ala Val Ile Ala 1. 5 1O 15 Gly Leu Pro Ser Thr Ala His Ala 2O
<210s, SEQ ID NO 8 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide
<4 OOs, SEQUENCE: 8 Met Ser Cys Thr Arg Ala Phe Llys Pro Lieu. Lieu. Lieu. Ile Gly Lieu Ala 1. 5 1O 15 Thr Lieu Met Cys Ser His Ala Phe Ala 2O 25
<210s, SEQ ID NO 9 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide
<4 OOs, SEQUENCE: 9 Met Arg Lieu Ala Ala Lieu Pro Lieu Lieu Lieu Ala Pro Leu Phe Ile Ala 1. 5 1O 15
Pro Met Ala Wall Ala 2O
<210s, SEQ ID NO 10 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide
<4 OOs, SEQUENCE: 10 Met Lys Lieu Lys Arg Lieu Met Ala Ala Met Thr Phe Val Ala Ala Gly 1. 5 1O 15
Wall Ala Thr Wall Asn Ala Wall Ala 2O
<210s, SEQ ID NO 11 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide
<4 OOs, SEQUENCE: 11 Met Arg Lieu. Thr Glin Ile Ile Ala Ala Ala Ala Ile Ala Lieu Val Ser 1. 5 1O 15
Thir Phe Ala Lieu. Ala
<210s, SEQ ID NO 12 &211s LENGTH: 21 US 8,906,636 B2 71 72 - Continued
212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide
<4 OOs, SEQUENCE: 12 Met Arg Asn Lieu. Lieu. Arg Gly Met Lieu Val Val Ile Cys Cys Met Ala 1. 5 1O 15 Gly Ile Ala Ala Ala 2O
<210s, SEQ ID NO 13 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide
<4 OOs, SEQUENCE: 13 Met Lys Lieu Lys Arg Lieu Met Ala Ala Met Thr Phe Val Ala Ala Gly 1. 5 1O 15
Wall Ala Thir Ala Asn Ala Wall Ala 2O
<210s, SEQ ID NO 14 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial Sequence & 22 O FEATURE; <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide
<4 OOs, SEQUENCE: 14 Met Glin Asn Tyr Llys Llys Phe Lieu. Lieu Ala Ala Ala Val Ser Met Ala 1. 5 1O 15
Phe Ser Ala Thir Ala Met Ala 2O
<210s, SEQ ID NO 15 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide
<4 OOs, SEQUENCE: 15 Met Pro Pro Arg Ser Ile Ala Ala Cys Lieu. Gly Lieu. Lieu. Gly Lieu. Lieu. 1. 5 1O 15
Met Ala Thr Glin Ala Ala Ala 2O
<210s, SEQ ID NO 16 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide
<4 OOs, SEQUENCE: 16 Met Lys Llys Ser Thr Lieu Ala Val Ala Val Thir Lieu. Gly Ala Ile Ala 1. 5 1O 15 US 8,906,636 B2 73 74 - Continued
Glin Glin Ala Gly Ala 2O
<210s, SEQ ID NO 17 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide
<4 OOs, SEQUENCE: 17 Met Lys Lieu Lys Arg Lieu Met Ala Ala Met Thr Phe Val Ala Ala Gly 1. 5 1O 15
Wall Ala Thir Ala Asn Ala Wall Ala 2O
<210s, SEQ ID NO 18 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide
<4 OOs, SEQUENCE: 18 Met Llys Phe Lys Glin Lieu Met Ala Met Ala Lieu Lleu Lieu Ala Lieu. Ser 1. 5 1O 15
Ala Wall Ala Glin Ala 2O
<210s, SEQ ID NO 19 &211s LENGTH: 33 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide
<4 OOs, SEQUENCE: 19 Met Glin Asn Arg Thr Val Glu Ile Gly Val Gly Lieu. Phe Lieu. Lieu Ala 1. 5 1O 15 Gly Ile Lieu Ala Lieu Lleu Lleu Lieu Ala Lieu. Arg Val Ser Gly Lieu. Ser 2O 25 3O
Ala
<210s, SEQ ID NO 2 O &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Corynebacterium diphtheriae 22 Os. FEATURE: <221s NAME/KEY: MOD RES <222s. LOCATION: (9) ... (9) <223> OTHER INFORMATION: Any amino acid
<4 OOs, SEQUENCE: 2O Met Ser Arg Llys Lieu. Phe Ala Ser Xaa Lieu. Ile Gly Ala Lieu. Lieu. Gly 1. 5 1O 15
Ile Gly Ala Pro Pro Ser Ala His Ala 2O 25
<210s, SEQ ID NO 21 &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Corynebacterium diphtheriae US 8,906,636 B2 75 76 - Continued
< 4 OOs SEQUENCE: 21 Gly Ala Asp Asp 1.
SEQ ID NO 22 LENGTH: 102 TYPE PRT ORGANISM: Wibrio cholerae
< 4 OOs SEQUENCE: 22
Thr Pro Glin Asn Ile Thr Asp Lieu. Cys Ala Glu Tyr His Asn Thr Gin 1. 15
Ile His Thr Lieu. Asn Asp Llys Ile Phe Ser Tyr Thir Glu Ser Lieu Ala 2O 25 3O
Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Asn Gly Ala Thir Phe 35 4 O 45
Glin Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Glin Lys Ala SO 55 6 O
Ile Glu Arg Met Lys Asp Thir Lieu. Arg Ile Ala Lell Thir Glu Ala 65 70 7s
Llys Val Glu Lys Lieu. Cys Val Trp Asn. Asn Lys Thir Pro His Ala Ile 85 90 95
Ala Ala Ile Ser Met Ala
SEQ ID NO 23 LENGTH: 306 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic polynucleotide FEATURE: NAME/KEY: CDS LOCATION: (1) ... (306)
< 4 OOs SEQUENCE: 23 acg ccg caa aat atc acc gac Ctg to go a gaa tat CaC aat ac C. Cala 48 Thr Pro Glin Asn Ile Thr Asp Lieu. Cys Ala Glu Tyr His Asn Thr Gin 1. 15 atc cat act ctd aac gac aaa at c titc agc a CC gag agc Ctg gct 96 Ile His Thr Lieu. Asn Asp Llys Ile Phe Ser Thir Glu Ser Lieu Ala 2O 25 3O ggc aag cqc gag atg gcg atc att acg tt C aac ggit gcg acc titt 144 Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Asn Gly Ala Thir Phe 35 4 O 45
Cag gtg gala gt C C cc ggc agt cag cac at C gat t cc Cag a.a.a. aag gcc 192 Glin Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Glin Lys Lys Ala SO 55 6 O att gala C9g atg aag gac acc Ct c cqt at C gcc tac ttg acc gala gCC 24 O Ile Glu Arg Met Lys Asp Thir Lieu. Arg Ile Ala Lell Thir Glu Ala 65 70 7s 8O aag gtg gag aag Ctg to gtt tog aac aac a.a.a. a CC cc.g CaC gcc atc 288 Llys Val Glu Lys Lieu. Cys Val Trp Asn. Asn Lys Thir Pro His Ala Ile 85 90 95 gcg gCC at C tog atg gcc 3 O 6 Ala Ala Ile Ser Met Ala
SEQ ID NO 24