(12) United States Patent (10) Patent No.: US 8,455,218 B2

Home , Autolysin, Carboxypeptidase A, Deuterolysin, Lysyl endopeptidase, Metalloendopeptidase, Plasmepsin

USOO84.55218B2

(12) UnitedO States Patent (10) Patent No.: US 8,455,218 B2 Jin et al. (45) Date of Patent: Jun. 4, 2013

(54) METHODS FOR G-CSF PRODUCTION INA EP 0207459 1, 1987 PSEUDOMONAS HOST CELL EP O243153 10, 1987 EP O2727O3 12/1987 EP O331186 9, 1989 (75) Inventors: Hongfan Jin, San Diego, CA (US); EP O335423 10, 1989 Lawrence Chew, San Diego, CA (US) EP O401384 12/1990 EP O459630 12/1991 (73) Assignee: Pfenex, Inc., San Diego, CA (US) EP O473268 3, 1992 WO WO90,12874 11, 1990 (*) Notice: Subject to any disclaimer, the term of this WO WO92,06116WO95/13393 4f19925, 1995 patent is extended or adjusted under 35 WO WO95/21254 8, 1995 U.S.C. 154(b) by 0 days. WO WO95/33057 12/1995 WO WO96,39422 12/1996 (21) Appl. No.: 13/076,315 WO WO99,58662 11, 1999 WO WOO1,73081 10, 2001 1-1. WO WO O2/2O766 3, 2002 (22) Filed: Mar. 30, 2011 WO WO O2/2O767 3, 2002 O O WO WO O2/O66514 8, 2002 (65) Prior Publication Data WO WO O2/O69232 9, 2002 WO WO O2/O77034 10, 2002 US 2011/0245474 A1 Oct. 6, 2011 WO WOO3,OO65O1 1, 2003 WO WOO3,O27288 4/2003 Related U.S. Application Data WO WO 03/031464 4/2003 WO WOO3,O76567 9, 2003 (60) Provisional application No. 61/320.239, filed on Apr. WO WO 2004/020576 3, 2004 1, 2010. WO WO-2007-107882 9, 2007 (51) Int. Cl. OTHER PUBLICATIONS CI2N 15/09 (2006.01) CI2N IS/10 (2006.01) Squires et al., Bioprocess International, 2004, 54-56, 58-59.* CI2P2/06 (200 6. 01) Bergey’s Manual of Determinative Bacteriology, R.E. Buchanan and N. E. Gibbons eds., pp. 217-289, 8' ed., The Williams & Wilkins Co., (52) U.S. Cl. Baltimore, MD, 1974. USPC ...... 435/69.1; 435/69.5; 435/71.2 Choi et al., “Development and optimization of two-stage cyclic fed (58) Field of Classification Search batch culture for hCG-CSF production using L-arabinose promoter of None Escherichia coli,” Bioprocess and Biosystems Engineering 24 (2001) See application file for complete search history. 51-58. Choi et al., “Plasmid Stability in Long-Term hCG-CSF Production (56) References Cited Using L-Arabinose Promoter System of Escherichia coli,” J. Microbiol. Biotechnol. (2000) 10(3), 321-326. U.S. PATENT DOCUMENTS Chung et al., “Overproduction of Human Granulocyte-Cology 4551.433. A 11, 1985 DeB Stimulating Factor Fused to the PelB Signal Peptide in Escherichia - - - SOC coli,”ss Journ. of Fermentation and Bioengineering, vol. 85, No. 4. 4,695.455 A 9, 1987 Barnes et al. 443-446 (1998). 4,755.465 A 7/1988 Gray et al. 4,810,643 A 3, 1989 Souza Cusi M. Grazia and D. Ferrero, Harlequin granulocyte-colony stimu 4,861,595 A 8, 1989 Barnes et al. lating factor interleukin 6 molecules with bifunctional and antago 4,904,584 A 2, 1990 Shaw nistic activities, Immunotechnology JID-951 19793 (1):61-69, 1997. 5,055,294 A 10, 1991 Gilroy Davis and Mingioli, “Mutants of Escherichia coli requiring 5,128,130 A 7/1992 Gilroy et al. methionine or Vitamin B12.” Bact 60:17-28 (1950). 5,169,760 A 12, 1992 Wilcox 5,281,532 A 1/1994 Rammler et al. (Continued) 5,508,192 A 4, 1996 Georgiou et al. 5,824,778 A 10, 1998 Ishikawa et al. 5,824,784. A 10, 1998 Kinstler et al. 7,001,892 B1* 2/2006 Chmielewski et al...... slass Primary Examiner Gyan Chandra 7,070,989 B2 7/2006 Lee et al. (74) Attorney, Agent, or Firm — Wilson, Sonsini, Goodrich 7,618,799 B2 * 1 1/2009 Coleman et al...... 435/183 & Rosati 2003/0167531 A1* 9, 2003 Russell et al...... 800,288 2005/0283000 A1 12/2005 Menart et al. 2006, OOO8877 A1 1/2006 Retallack et al. 2006,0040352 A1 2, 2006 Retallack et al. (57) ABSTRACT 58,856 A. 358; St..1. The present invention relates to the field of recombinant pro 2008.0193974 A1* 8, 2008 Coleman et al...... 435/69.1 tein production in bacterial hosts. It further relates to expres 2008/0269070 A1* 10, 2008 Ramseier et al...... 506,14 sion of soluble, active recombinant protein by using secretion 2009/0092582 A1 4, 2009 Bogin et al. signals to direct the protein to the periplasmic space of a 2009/0275518 A1 11/2009 Gonthier et al. 2009/0325230 A1 12, 2009 Schneider et al. bacterial cell. In particular, the present invention relates to a 2010.0137162 A1 6, 2010 Retallack et al. production process for obtaining soluble hC-CSF protein from a bacterial host. FOREIGN PATENT DOCUMENTS AU T638O, 91 11, 1991 AU 10948.92 8, 1992 26 Claims, 8 Drawing Sheets US 8,455,218 B2 Page 2

OTHER PUBLICATIONS Welch, et al., 2009, PLoS One, “Design Parameters to Control Syn Devlin et al., “Alteration of amino-terminal codons of human granu thetic Gene Expression in Escherichia coli,” 4(9): e7002. locyte-colony-stimulating factor increases expression levels and Weston, B., Todd, R. F., 3rd, Axtell, R., Balazovich, K., Stewart, J., Locey, B.J., Mayo-Bond, L., Loos, P. Hutchinson, R. & Boxer, L. A. allows efficient processing by methionine aminopeptidase in (1991). Severe congenital neutropenia: clinical effects and neutrophil Escherichia coli,” Gene, 65 (1988) 13-22. function during treatment with granulocyte colony-stimulating fac Frishman et al., “Starts of Bacterial Genes: Estimating the Reliability tor. J Lab Clin Med 117, 282-90. of Computer Predictions.” Gene 234(20):257-265 (1999). Wingfield et al., Characterization of recombinant-derived granulo Ghane, et al., “Over Expression of Biologically Active Interferon cyte-colony stimulating factor (G-CSF), Biochem. J. (1988) 256, Beta Using Synthetic Gene in E. coli,” Journ. of Sciences, Islamic 213-218. Republic of Iran 19(3): 203-209 (2008). Zaveckas et al., “Effect of Surface histidine mutations and their Hong et al., “Production of biologically active hG-CSF by transgenic number on the partitioning and refolding of recombinant human granulocyte-colony Stimulating factor (CyS 17 Ser) in acqueous two plant cell suspension culture.” Enzyme and Microbial Technology 30 phase systems containing chelated metal ions.” Journ. of Chroma (2002) 763-767. tography B, 852 (2007) 409-419. Ikehata et al., “Primary structure of nitrile hydratase deduced from Yoon et al., “Secretory Production of Recombinant Proteins in the nucleotide sequence of a Rhodococcus species and its expression Escherichia coli, ' Recent Patents on Biotechnology, 4, 23-29 in Escherichia coli.” Eur J. Biochem 181(3):563-570 (1989). (2010). Krishna R. et al., (2008) Mol Biotechnology "Optimization of the PCT/US2011/030593 International Search Report dated Dec. 23, AT-content of Codons Immediately Downstream of the Initiation 2011. Codon and Evaluation of Culture Conditions for High-level Expres Botos et al., “The Catalytic Domain of Escherichia coli Lon Protease sion of Recombinant Human G-CSF in Escherichia coli,' 38:221 Has a Unique Fold and a Ser-Lys Dyad in the Active Site.” 2004. The 232. Journal of Biological Chemistry, vol. 279, No. 9: 8140-8148. Jeong et al., “Secretory Production of Human Granulocyte Colony Covalt J.C.et al., “Temperature, media, and point of induction affect Stimulating Factor in Escherichia coli.” Protein Expression and Puri the N-terminal processing of interleukin-1beta.” Protein Expr Purif fication 23, 311-318 (2001). 41: 45-52 (2005). Jevsevar et al., “Production of Nonclassical Inclusion Bodies from Gilbert et al., “A New Cell Surface Proteinase: Sequencing and Which Correctly Folded Protein Can Be Extracted.” Biotechnol. P. Analysis of the priB Gene from Lactobacillus delbrueckii subsp. (2005) 21, 632-639. Bulgaricus,” Journal of Bacteriology, 1996, 178(11): 3059-3065. Kang et al., “High Level Expression and Simple Purification of Hammerling et al., “In Vitro bioassay with enhanced sensitivity for Recombinant Human Granulocyte Colony-Stimulating Factor in E. human granulocyte colony-stimulating factor.” J. Pharm. Biomed. coli,” Biotechnology Letters, vol. 17, No. 7 (1995) 687-692. Anal. 3 (1995) 9-20. Neupogen (Filgrastim) Prescription Drug Product Insert (Sep. 2007). Herman et al., “Characterization, formulation and stability of Perez-Perez et al., “DNAKDNAJ Supplementation Improves the Neupogen (Filgrastim), a recombinant human granulocyte-colony Periplasmic Production of Human Granulocyte-Colony Stimulating stimulation factor.” Pharmaceutical Biotechnology, edited by Pearl Factor in Escherichia coli,” Biochemical and Biophysical Research man and Wang, vol. 9, (1996) 303-28. Communications, vol. 210, No. 2, 1995, pp. 524-529. Jin et al., “Soluble periplasmic production of human granulocyte Rao et al., “Optimization of the AT-content of Codons Immediately colony-stimulating factor (G-CSF) in Pseudomonas fluorescens.” Downstream of the Initiation Codon and Evaluation of Culture Con Protein Expression and Purification, vol. 78(1), (2011): 69-77. ditions for High-level Epression of Recombinant Human G-CSF in Keiler, K.C. & Sauer, R.T., “Identification of Active site Residues of Escherichia coli.” Mol. Biotechnol (2008) 38:221-232. the Tsp Protease.” The Journal of Biological Chemistry, 1995, vol. Riesenberg, D et al., 1991, “High cell density cultivation of 270, No. 48, pp. 28864-28868. Escherichia coli at controlled specific growth rate.” J. Biotechnol. 20 Kramer, Irene, Ph.D., “Recombinant G-CSF products and what phar (1):17-27. macists need to know.” European Journal of Hospital Pharmacists, J. Sanchez-Romero & V. De Lorenzo (1999) Manual of Industrial May 2011, vol. 17, pp. 36-45. Microbiology and Biotechnology (A. Demain & J. Davies, eds.) pp. Louis et al., “Specificity of Pseudomonas aeruginosa Serralysin 460-74 (ASM Press, Washington, D.C.). revisited using biologically active peptides as Substrates.” Schneider, et al., 2005, "Auxotrophic markers pyrF and proC can Biochimica et Biophysica Acta, 1998, pp. 378-386. replace antibiotic markers on protein production plasmids in high Okabe, et al., “In vitro and in vivo hematopoietic effect of mutant cell-density Pseudomonas fluorescens fermentation.” Biotechnol. human granulocyte colony-stimulating factor.” Blood, 75. (1990), Progress 21(2): 343-8. pp. 1788-1793. Schweizer, “Vectors to express foreign genes and techniques to moni Pallen, M.J. & Wren, B.W. “The Htr A Family of serine proteases.” tor gene expression in Pseudomondas.” Curr Op Biotech 12:439-445 Molecular Microbiology, 1997, 26(2), pp. 209-221. (2001). Patterson-Ward et al., “Detection and characterization of two ATP R. Slater & R. Williams, Molecular Biology and Biotechnology, J. dependent conformational changes in proteolytically inactive Walker & R. Rapley, eds., pp. 125-154. The Royal Society of Chem Escherichia coli Lon mutants by stopped flow kinetic techniques.” istry, Cambridge, UK, 2000. Biochemistry, 2007, 46(47): 13593-13605 doi:10.1021/bi701649b. R. Sorg, J. Enczmann, U. Sorg, K. Heermeier, E. M. Schneider, and Retallack et al., “Transport of heterologous proteins to the P. Wernet. Rapid and sensitive mRNA phenotyping for interleukins periplasmic space of Pseudomonas fluorescens using a variety of (IL-1 to IL-6) and colony-stimulating factors (G-CSF, M-CSF, and native signal sequences.” Biotechnol Lett 29, (2007), pp. 1483-1491. GM-CSF) by reverse transcription and Subsequent polymerase chain Sakoh et al., “Proteolytic Activity of HtpX, a Membrane-bound and reaction, Exp. Hematol JID-0402313 19 (9):882-887, 1991. Stress-controlled Protease from Escherichia coli, 2005, Journal of B. E. Suzek et al., “A probabilistic method for identifying start Biological Chemistry, vol. 280, No. 39, pp. 33305-33310. codons in bacterial genomes.” Bioinformatics 17(12): 1123-30 (Dec. Tanaka et al., “Three types of recombinant human granulocyte 2001). colony-stimulating factor have equivalent biological activities in Vanz et al., “Human Granulocyte Colony Stimulating factor (hG monkeys.” Cytokine 9, (1997), pp. 360-369. CSF): cloning, overexpression, purification and characterization.” Microbial Cell Factories 2008, 7:13. * cited by examiner U.S. Patent Jun. 4, 2013 Sheet 1 of 8 US 8,455,218 B2

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(luu/fid)uo??euqueouooJeonpul 8CHRI[15)I,H. US 8,455,218 B2 1. 2 METHODS FOR G-CSF PRODUCTION INA embodiments, the expression construct is a plasmid. In other PSEUDOMONAS HOST CELL embodiments, the expression construct comprises sequence encoding G-CSF protein fused to a secretion signal. In certain CLAIM OF PRIORITY embodiments, the Secretion signal directs transfer of the G-CSF protein to the periplasm in the Pseudomonas host cell. This application claims priority under 35 U.S.C. S 119(e) to In certain embodiments, the Secretion signal is cleaved from U.S. Provisional Application Ser. No. 61/320,239, filed Apr. said G-CSF protein in said Pseudomonad host cell. In certain 1, 2010. The content of this application is hereby incorporated embodiments, the secretion signal protein sequence com by reference in its entirety. prises any one of SEQID NOs: 8-26. In certain embodiments, 10 at least 50% of said G-CSF protein is expressed in the soluble SEQUENCE LISTING fraction. In embodiments, the protease is a serine protease. In The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated embodiments, the serine protease is PrtB, and its gene is prtB. 15 In other embodiments, the mutation is a complete deletion. by reference in its entirety. Said ASCII copy, created on Sep. In embodiments, the Pseudomonas host cell is a 7, 2012, is named 38194201.txt and is 27,653 bytes in size. Pseudomonas host cell. In certain embodiments, the BACKGROUND OF THE INVENTION Pseudomonas host cell is a Pseudomonas fluorescens host cell. Human granulocyte colony-stimulating factor (hG-CSF) is In embodiments, the G-CSF protein is human G-CSF pro a cytokine that can play a role in the proliferation and differ tein. In embodiments, the yield of said G-CSF protein is about entiation of hemopoietic precursor cells and the activation of 0.1 g/L to 10 g/L. In embodiments, the G-CSF protein is mature neutrophilic granulocytes. Recombinant hCG-CSF can active. In embodiments, the activity is determined by binding be used, for example, as an injectable to i) selectively stimu recombinant G-CSF receptor. late the growth of white blood cells, ii) to help reduce the 25 The invention further includes a method comprising pro incidence of infection in patients undergoing certain cancer ducing a G-CSF protein in a Pseudomonas host cell, wherein chemotherapy, iii) for the mobilization of peripheral blood the G-CSF comprises an N-terminal methionine, and the progenitor cells, and iv) for the treatment of severe chronic yield of Met-G-CSF protein is about 0.1 g/L to 10 g/L. In neutropenia. embodiments, the method comprises expressing said G-CSF Two forms of recombinant hC-CSF are currently available 30 protein from an expression construct. In other embodiments, for clinical use on the market: a glycosylated form obtained by expression in mammalian cells and a non-glycosylated said expression construct is a plasmid. In certain embodi form synthesized in an E. coli expression system. Economical ments, the expression construct comprises a sequence encod large-scale production of recombinanthC-CSF is still a great ing the G-CSF protein fused to a secretion signal. In embodi challenge with respect to biosynthesis and downstream pro 35 ments, the secretion signal directs transfer of the G-CSF cessing because the expression efficiency of a hO-CSF gene protein to the periplasm in the Pseudomonas host cell. In in the E. coli expression system is low and overexpression certain embodiments, the secretion signal is cleaved from generally results in partitioning of the expressed protein as said G-CSF protein in said Pseudomonas host cell. In certain insoluble material in inclusion bodies. Expression of hC-CSF embodiments, the secretion signal protein sequence com in insoluble inclusion bodies (IBS) can require lengthy down 40 prises any one of SEQID NOs: 8-26. In certain embodiments, stream process to solublize and refold the target protein. The at least 50% of said G-CSF protein is expressed in the soluble available periplasmically expressed hC-CSF products either fraction. lack the N-terminal methionine or have alternative N-termi In embodiments, the Pseudomonas host cell is a nal sequence compared to the drug Filgrastim. There has been Pseudomonas host cell. In certain embodiments, the work reported in E. coli relating to secretion of hG-CSF in a 45 Pseudomonas host cell is a Pseudomonas fluorescens host soluble form; however, in these expression systems a peptide cell. tag was added and tag removal upon purification required. In embodiments, the G-CSF protein is human G-CSF pro Thus, there is a need for new methods of expressing hC-CSF tein. In certain embodiments, the G-CSF protein is active. In comprising an N-terminal methionine (Met-G-CSF) and no other embodiments, the activity is determined by binding Sequence tag. 50 recombinant G-CSF receptor. The present invention also provides a composition com SUMMARY OF THE INVENTION prising G-CSF protein obtained according to the methods The present invention relates to the expression of recom described herein. In embodiments, the G-CSF protein com binant human G-CSF protein fused to a secretion signal in a 55 prises an N-terminal methionine. In embodiments, the recom Pseudomonad host cell, wherein the recombinant human binant toxin protein is produced in a strain of Pfluorescens G-CSF protein can be directed to the periplasm of the identified herein as producing a high yield of the soluble Pseudomonad host cell, and soluble recombinant human protein and/or a high yield of GCSF comprising the N-termi G-CSF can be generated that lacks the secretion signal and nal methionine. In certain embodiments, the recombinant comprises and N-terminal methionine (Met-G-CSF). 60 GCSF protein is produced in a strain of P. fluorescens In particular, the present invention provides a method com described herein as producing the highest yield of desirable prising producing a G-CSF protein in a Pseudomonad host GCSF protein. In other embodiments, the recombinant pro cell, wherein the G-CSF comprises an N-terminal methion tein is produced in a strain of Pfluorescens described herein ine, and wherein said Pseudomonad host cell comprises a as one used for fermentation production of the GCSF protein. mutation in a gene expressing a protease. 65 In specific embodiments, the recombinant protein is pro In embodiments, the producing comprises expressing said duced in a strain of P. fluorescens having arxfo8627 deletion, G-CSF protein from an expression construct. In certain and further wherein the G-CSF protein is expressed from an US 8,455,218 B2 3 4 expression construct comprising the sequence encoding the at 450 nm representing cell proliferation is shown on the G-CSF protein fused to a DsbA secretion signal. Y-axis. The error bars represent the standard error of three replicates for each point. INCORPORATION BY REFERENCE DETAILED DESCRIPTION OF THE INVENTION All publications, patents, and patent applications men tioned in this specification are herein incorporated by refer Overview ence to the same extent as if each individual publication, The present invention relates to methods for producing patent, or patent application was specifically and individually soluble recombinant human granulocyte colony-stimulating indicated to be incorporated by reference. 10 factor (hG-CSF) in a Pseudomonas host cell. High levels of expression of recombinant hCG-CSF can be achieved, and the BRIEF DESCRIPTION OF THE DRAWINGS hG-CSF protein can be prepared to a high level of purity. The host cell can be Pseudomonas fluorescens. The codons in a The novel features of the invention are set forth with par construct used to express hCG-CSF can be selected to be opti ticularity in the appended claims. A better understanding of 15 mized for expression of hCG-CSF in the host strain used, e.g., the features and advantages of the present invention will be Pseudomonas fluorescens. obtained by reference to the following detailed description Nucleic acid constructs can encode hCG-CSF fused to any of that sets forth illustrative embodiments, in which the prin a selection of native secretion leaders, e.g., secretion leaders ciples of the invention are utilized, and the accompanying native to Pfluorescens. Any of a variety genetic backgrounds drawings of which: of Pseudomonas stains, e.g., Pfluorescens host strains, can FIG. 1 illustrates expression analysis of the Met-G-CSF be used. In one embodiment, the genetic background com with 19 different secretion leaders. One-way analysis (quan prises a mutation in one or more genes that encode proteases. tiles) of soluble volumetric G-CSF expression, as quantified In another embodiment, the protease is prtB. by BLI interferometry, using JMP Software (Cary, N.C.). 25 In one embodiment, the secretion leader can transport Each bar represents results from a single expression strain, soluble hG-CSF to the periplasm. In other embodiments, the and yields for each of three replicates are represented by black purification of hC-CSF does not require solubilization and dots. The expression level for all strains is shown. For conve Subsequent refolding. In other embodiments, at least a portion nience, the periplasmic Met-G-CSF constructs are repre of hCG-CSF is not expressed in inclusion bodies. In other sented by the secretion leaders. 30 embodiments, recombinant hCG-CSF is expressed devoid of FIG. 2 illustrates intact mass analysis of target protein any peptide tag for purification and therefore does not require produced from representative strains following expression in additional processing upon purification. In other embodi minibioreactor cultures. The deconvoluted intact mass of the ments, the secretion leader is efficiently processed from the main peak corresponds to the intact Met-G-CSF at 18.80 kDa solubly expressed hC-CSF and the protein contains an N-ter from CS529-901(A). Strain CS529-712 also produced a 35 minal methionine (Met-G-CSF), the same as the drug minor peak representing the des-Met product at 18.67 kDa Filgrastim. In other embodiments, the expressed protein is (B). non-glycosylated, as is hC-CSF produced in Ecoli. In other FIG.3 depicts a representative expression plasmid map for embodiments, an expression plasmid for periplasmic produc a plasmid containing a G-CSF gene for expression in Pfluo tion of hC-CSF does not utilize any antibiotic resistance 40 marker gene for selection and maintenance, thus eliminating rescens. Plasmid p529-016 encodes G-CSF with Dsba secre complicated processes for Subsequent removal of plasmid tion signal. Open reading frames are indicated by arrows, DNA required for production of biopharmaceuticals. In other including the pyrF selectable marker. The relevant promoter embodiments, fermentation conditions are scalable for large and transcriptional terminator elements are also annotated. volume production. The methods of the provided invention FIG. 4 depicts soluble Met G-CSF production for replicate 45 can yield high levels of soluble, active hC-CSF protein with fermentations of strain CS529-901. Replicate fermentations an N-terminal methionine (Met-G-CSF). resulted in consistent Met G-CSF production at 0.35 g/L as G-CSF determined by BLI binding assay, with time post induction Granulocyte colony-stimulating factor (also known as shown on the X-axis and yield of active protein shown on the G-CSF, GCSF, colony-stimulating factor 3, CSF3, or Y-axis. Error bars indicate standard deivation from 3 replicate 50 CSF3OS) is a colony-stimulating factor hormone. In humans, samples for each time point. G-CSF can be produced by a number of different tissues. FIG. 5 depicts a gel-like image of soluble Met G-CSF G-CSF can be secreted by monocytes, macrophages, neutro production in replicate fermentations of strain CS529-901. phils, stromal cells, fibroblasts, and endothelial cells. Synthe Time post-induction for each fermentation is indicated above sis of G-CSF can be induced by bacterial endotoxins, TNF, each lane. The migration of induced recombinant protein is 55 IL1, IL17, and GM-CSF (granulocyte-macrophage colony indicated by the red arrow. stimulating factor). Prostaglandin E2 can inhibit the synthesis FIG. 6 shows an exemplary DNA sequence encoding of G-CSF. hGCSF that has been optimized for expression in Pfluore G-CSF can serve as a growth factor or cytokine that can scens (SEQ ID NO: 27), and the corresponding amino acid stimulate the bone marrow to produce granulocytes and stem sequence (SEQID NO: 28). 60 cells and release them into the blood. G-CSF can also stimu FIG. 7 shows the h-GCSF amino acid sequence (SEQ ID late the survival, proliferation, differentiation, and function of NO: 28). neutrophil precursors and mature neutrophils. G-CSF can FIG. 8 shows the activity of P fluorescens-produced regulate these cells using Janus kinase (JAK)/signal trans G-CSF as indicated by proliferation of the murine myeloblas ducer and activator of transcription (STAT) and Ras/mitogen tic cell line NFS-60. The concentration of CS5329-901-pro 65 activated protein (MAP) kinase and phosphatidylinositol duced G-CSF (open circles) and Neupogen R. (closed 3-kinase (PI3K)/protein kinase B (Akt) signal transduction squares) in pg/ml is shown on the X-axis, and the absorbance pathways. US 8,455,218 B2 5 6 DNA their activity by mutagenizing specific residues and linking The human G-CSF gene maps to chromosome 17q21-q22 non-peptide moieties such as PEG molecules (e.g., PCT and is located in the vicinity of a translocation break point Application Publication Nos. WO 03/031464 and WO which can occur in acute promyelocytic leukemias. The 03/006501; EP401384, EP473268, EP335423, and U.S. Pat. human G-CSF gene contains 5 exons and 4 introns. G-CSF Nos. 5,824,778 and 5,824,784), all incorporated herein by mRNA can be differentially spliced, leading to the production reference in their entirety. Antibodies against human G-CSF of two variant forms of the protein, one shortened by 3 amino have been described (e.g., EP0331186). acids. The mouse orthologue of CSF3 HUMAN is CSF3 Recombinant G-CSF MOUSE (Swissprot databank) and is found on mouse chro Recombinant G-CSF is marketed under the generic names mosome 11. The genomic organization of the mouse gene is 10 Filgrastim and Lenograstim and under the brand names similar to that of the human (5 exons and 4 introns). Neupogen, Neutrogin, and Granocyte. Filgrastim is the Protein generic name for a non-glycosylated recombinant G-CSF In human cells, two different polypeptides of G-CSF of (brand name Neupogen; Amgen Inc., Thousand Oaks, Calif.). molecular weight 19,600 can be synthesized from the same Filgrastim is a 175-amino acid protein produced by a gene gene by differential mRNA splicing (Nagata et al., 1986: 15 encoding hCG-CSF in E. coli. Filgrastim has an N-terminal Souza et al., 1986; Metcalf, 1985). The G-CSF gene can methionine. Filgrastimalso differs from the form of hC-CSF encode a protein of 207 amino acids containing a hydropho expressed in a human cell because Filgrastim lacks glycosy bic secretory signal sequence of 30 amino acids. Swissprot lation. Lenograstim (brand name Graslopin, Granocyte), databank lists a 207 amino acid form of G-CSF (P09919-1) developed by Ligand Pharmaceuticals, is a glycosylated and a 204 amino acid form (the “isoform short, P09919-2). recombinant form of human granulocyte colony stimulating Mature forms of the two polypeptides differ by the presence factor. Neulasta (pegfilgrastim) is obtained by attaching a 20 (long form) or absence (shortform) of 3 amino acids (177 and kDa monomethoxypolyethylene glycol to the N-terminal 174 amino acids, respectively). Both the long and short form methionyl residue of Filgrastim (pegylated Filgrastim). can have G-CSF biological activity. Filgrastim can be administered to patients receiving G-CSF contains 5 cysteine residues, four of which can 25 myelosuppressive chemotherapy, patients with acute myeloid form disulfide bonds (between amino acids 36 and 42 and leukemia that receive induction or consolidation chemo between amino acids 64 and 74) (numbered with respect to therapy, patients receiving bone marrow transplant, patients the mature (lacking the signal peptide) short form of the undergoing peripheral blood progenitor cell collection and polypeptide). A free cysteine can be found at position 17. An therapy, or patients with severe chronic neutropenia. O-glycosylation site can occur at Thr-133 in G-CSF. The 30 A recombinant mutated form of human G-CSF is protein can be glycosylated with O-glycan consisting of Gal KW-2228 (Marograstim and Nartograstim), an N-terminally GalNAc disaccharide, which can be modified with up to two modified form of human G-CSF in which Thr1, Leu3, Gly4, sialic acid residues (which occurs in recombinantly expressed Pro5, and Cys 17 are substituted with Ala, Thr, Tyr, Arg, and G-CSF from CHO cells). The sugar moiety of G-CSF is not Ser, respectively. This protein can be produced in Escherichia required for full biological activity. The biologically active 35 coli. form is a monomer. Modifications Human G-CSF has four stretches of helices: between resi In some embodiments, modified versions of hCG-CSF can dues 11 and 41 (helix A), 71 and 95 (helix B), 102 and 125 be generated. In general, with respect to an amino acid (helix C), and 145 and 170 (helix D). A left-handed four-helix sequence, the term “modification' includes Substitutions, bundle can beformed, withhelices A and Baligned parallel to 40 insertions, elongations, deletions, and derivatizations alone one another (up-up) and antiparallel to helices C and D or in combination. In some embodiments, the peptides may (down-down). Part of the AB loop connecting helices A and B include one or more modifications of a “non-essential amino has an additional short fifth helix (E). G-CSF has a pl of 5.5. acid residue. In this context, a "non-essential amino acid Sequence variants of hC-CSF are described, for example, residue is a residue that can be altered, e.g., deleted or Sub in U.S. Patent Application No. 2009/0275518, which is incor 45 stituted, in the novel amino acid sequence without abolishing porated herein in its entirety. hCG-CSF and related mutants or or Substantially reducing the activity (e.g., the agonist activ variant cDNAs and proteins have been disclosed in ity) of the peptide (e.g., the analog peptide). In some embodi EP0243153. Splicing variants of G-CSF have been reported ments, the peptides may include one or more modifications of in R. Sorg, J. Enczmann, U. Sorg, K. Heermeier, E. M. an “essential amino acid residue. In this context, an “essen Schneider, and P. Wernet. Rapid and sensitive mRNA pheno 50 tial amino acid residue is a residue that when altered, e.g., typing for interleukins (IL-1 to IL-6) and colony-stimulating deleted or Substituted, in the novel amino acid sequence the factors (G-CSF, M-CSF, and GM-CSF) by reverse transcrip activity of the reference peptide is substantially reduced or tion and Subsequent polymerase chain reaction, Exp Hematol abolished. In such embodiments where an essential amino JID-0402313 19 (9):882-887, 1991; Cusi M. Grazia and D. acid residue is altered, the modified peptide may possess an Ferrero, Harlequin granulocyte-colony stimulating factor 55 activity of hC-CSF of interest in the methods provided. The interleukin 6 molecules with bifunctional and antagonistic substitutions, insertions and deletions may be at the N-termi activities, Immunotechnology JID-9511979 3 (1):61-69, nal or C-terminal end, or may be at internal portions of the 1997; and WO03027288A1. Analogs of human G-CSF have protein. By way of example, the protein may include 1, 2, 3, been generated by mutagenesis or by fusion with heterolo 4, 5, 6, 7, 8, 9, 10, or more substitutions, both in a consecutive gous sequences (e.g., PCT Application Nos. WO 04/020576; 60 manner or spaced throughout the peptide molecule. Alone or WO 02/020767; WO 02/020766; WO 02/066514; WO in combination with the Substitutions, the peptide may 02/077034; WO 03/076567; WO02/069232; WO 01/073081; include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertions, again WO99/58662: WO 96/39422: WO95/21254; WO95/13393; either in consecutive manner or spaced throughout the pep WO95/33057; WO 92/06116; WO 90/12874; EP272703; tide molecule. The peptide, alone or in combination with the EP459630; EP243153: U.S. Pat. No. 4,904,584: U.S. Pat. No. 65 Substitutions and/or insertions, may also include 1, 2, 3, 4, 5, 4,810,643; AU 76380/91; and AU 10948/92). Non-natural 6, 7, 8, 9, 10, or more deletions, again either in consecutive variants of human G-CSF have been generated to improve manner or spaced throughout the peptide molecule. The pep US 8,455,218 B2 7 8 tide, alone or in combination with the Substitutions, insertions Expression Systems and/or deletions, may also include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The methods of the provided invention can comprise or more amino acid additions. expressing recombinant hC-CSF from an expression con Substitutions include conservative amino acid substitu struct in a Pseudomonas host cell. The expression construct 5 can be, for example, a plasmid. In some embodiments, a tions. A "conservative amino acid substitution' is one in plasmid encoding hC-CSF sequence can comprise a selection which the amino acid residue is replaced with an amino acid marker, and host cells maintaining the plasmid can be grown residue having a similar side chain, or physicochemical char under selective conditions. In some embodiments, the plas acteristics (e.g., electrostatic, hydrogen bonding, isosteric, mid does not comprise a selection marker. In some embodi hydrophobic features). The amino acids may be naturally ments, the expression construct can be integrated into the host occurring or normatural (unnatural). Families of amino acid 10 cell genome. In some embodiments, the expression construct residues having similar side chains are known in the art. encodes hC-CSF fused to a secretory signal that can direct These families include amino acids with basic side chains hG-CSF to the periplasm. In some embodiments, the secetory (e.g. lysine, arginine, histidine), acidic side chains (e.g., signal can be cleaved in the host cell resulting in hC-CSF with aspartic acid, glutamic acid), uncharged polar side chains an N-terminal methionine (Met-G-CSF). 15 Methods for expressing heterologous proteins, including (e.g., glycine, asparagine, glutamine, serine, threonine, useful regulatory sequences (e.g., promoters, secretion lead tyrosine, methionine, cysteine), nonpolar side chains (e.g., ers, and ribosome binding sites), in Pseudomonas host cells, alanine, Valine, leucine, isoleucine, proline, phenylalanine, as well as host cells useful in the methods of the present tryptophan), B-branched side chains (e.g., threonine, Valine, invention, are described, e.g., in U.S. Pat. App. Pub. No. isoleucine) and aromatic side chains (e.g., tyrosine, phenyla 2008/0269070 and U.S. patent application Ser. No. 12/610, lanine, tryptophan, histidine). Substitutions may also include 207, both titled “Method for Rapidly Screening Microbial non-conservative changes. Hosts to Identify Certain Strains with Improved Yield and/or G-CSF Receptors Quality in the Expression of Heterologous Proteins. U.S. The G-CSF receptor (also known as CSF3R, CD114, or Pat. App. Pub. No. 2006/0040352, “Expression of Mamma GCSFR) is expressed on all cells of the neutrophil and granu 25 lian Proteins in Pseudomonas Fluorescens, and U.S. Pat. locyte lineage. It is expressed also in placenta cells, endothe App. Pub. No. 2006/0110747, “Process for Improved Protein lial cells and various carcinoma cell lines. The human recep Expression by Strain Engineering all incorporated herein by tor has a length of 813 amino acids. CSF3R contains an reference in their entirety. These publications also describe extracellular ligand-binding domain, a transmembrane bacterial host strains useful in practicing the methods of the domain, and a cytoplasmic domain. The human receptor 30 invention, that have been engineered to overexpress folding modulators or wherein protease mutations have been intro shows 62.5% sequence homology to the murine receptor. The duced, in order to increase heterologous protein expression. receptor can bind G-CSF with high affinity (K–550 Sequence leaders are described in detail in U.S. Patent App. picoM). The gene encoding the human G-CSF receptor Pub. No. 2008/0193974, “Bacterial leader sequences for (CSF3R) maps to chromosome 1 p32-p34. 3. 35 increased expression, and U.S. Pat. App. Pub. No. 2006/ At least four different forms of the human G-CSF receptor, 0008877, “Expression systems with Sec-secretion.” both resulting from alternative splicing of the mRNA, have been incorporated herein by reference in their entirety, as well as in cloned. Three of the isoforms can be membrane-bound and U.S. Pat. App. Ser. No. 12/610,207. the other can be secreted and soluble. One variant contains a U.S. Pat. App. Pub. No. 2008/0269070, incorporated deletion of the transmembrane region and is probably a 40 herein by reference, describes host cells having a mutation in soluble form of the receptor. Another variant contains 27 a protease gene expressing a protease, resulting in the inacti additional amino acids in its cytoplasmic domain. vation of the protease. Protease mutations described in U.S. Mutations in CSF3R are a cause of Kostmann syndrome, Pat. App. Pub. No. 2008/0269070 include, for example, a also known as severe congenital neutropenia. Severe con mutation of a gene from which a serine peptidase, an S41 genital neutropenia (Kostmann syndrome) is characterized 45 protease, an STE24 endopeptidase, or a lon protease is by profound absolute neutropenia and a maturation arrest of expressed. Inactivation of a protease by mutation in any of the marrow progenitor cells at the stage of promyelocytes and protease open reading frames provided hereinas SEQID NO: myelocytes. A somatic point mutation in one allele of the 29 (RXF01250, a serine peptidase), SEQ ID NO: 30 G-CSF receptor gene in a patient with severe congenital neu (RXF06586, an S41 protease), SEQID NO:31 (RXF05137, tropenia results in a cytoplasmic truncation of the receptor. 50 htpX, an STE24 endopeptidase), SEQ ID NO: 32 The mutant receptor chain can transduce a strong growth (RXF04653, alon protease), SEQID NO:33 (RXF08627.2, signal but this signal is unable to trigger maturation. The PrtB, an extracellular serine protease), and SEQID NO: 34 mutant receptor chain may act in a dominant negative manner (RXF04304.1, AprA, a serralysin) is contemplated. to block granulocytic maturation. Promoters G-CSF can form a tetrameric complex with GCSFR. The 55 The promoters used in accordance with the present inven complex can contain two ligand molecules and two receptor tion may be constitutive promoters or regulated promoters. molecules. The N-terminal region (residues 20-46) and the Common examples of useful regulated promoters include carboxy terminal region (including helix D) of G-CSF can be those of the family derived from the lac promoter (i.e. the lacz involved in binding to the receptor. One receptor-binding site promoter), especially the tac and trc promoters described in involves various residues on the helices A and C. A second 60 U.S. Pat. No. 4,551,433 to DeBoer, as well as Ptac16, Ptac17, binding site may be located on the helix E (1997, PMID PtacII, PlacUV5, and the T71ac promoter. In one embodi 9194183; 2003, PMID 12946100). A residue that plays a role ment, the promoter is not derived from the host cell organism. in receptor binding is Glu 19 (in helix A). Other residues that In certain embodiments, the promoter is derived from an E. can play functional roles are Lys 40, Glu 46 (helix E) and Phe coli organism. 144 (helix D). Val 48, Leu 49 (helix E), Leu15 (helix A), Asp 65 Inducible promoter sequences can be used to regulate 112 and Leu124 (helix C) appear to play a role in biological expression of hCG-CSF in accordance with the methods of the activity. invention. In embodiments, inducible promoters useful in the US 8,455,218 B2 10 methods of the present invention include those of the family tor compounds are classified as either inducers or co-repres derived from the lac promoter (i.e. the lacZ promoter), espe sors, and these compounds include native effector cially the tac and trc promoters described in U.S. Pat. No. compounds and gratuitous inducer compounds. Many regu 4,551,433 to DeBoer, as well as Ptac16, Ptac17, PtacII, lated-promoter/promoter-regulatory-protein/effector-com PlacUV5, and the T71ac promoter. In one embodiment, the pound trios are known in the art. Although an effector com promoter is not derived from the host cell organism. In certain pound can be used throughout the cell culture or embodiments, the promoter is derived from an E. coli organ fermentation, in a preferred embodiment in which a regulated ism. In some embodiments, a lac promoter is used to regulate promoter is used, after growth of a desired quantity or density expression of hCG-CSF from a plasmid. In the case of the lac of host cell biomass, an appropriate effector compound is 10 added to the culture to directly or indirectly result in expres promoter derivatives or family members, e.g., the tac pro sion of the desired gene(s) encoding the protein or polypep moter, an inducer is IPTG (isopropyl-B-D-1-thiogalactopyra tide of interest. noside, also called "isopropylthiogalactoside'). In certain In embodiments wherein a lac family promoter is utilized, embodiments, IPTG is added to culture to induce expression a lad gene can also be present in the system. The lad gene, of hC-CSF from a lac promoter in a Pseudomonas host cell. 15 which is normally a constitutively expressed gene, encodes Common examples of non-lac-type promoters useful in the Lac repressor protein Lad protein, which binds to the lac expression systems according to the present invention operator of lac family promoters. Thus, where a lac family include, e.g., those listed in Table 1. promoter is utilized, the lad gene can also be included and expressed in the expression system. TABLE 1. 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 Other Regulatory Elements PR High temperature In embodiments, soluble recombinant hC-CSF is present P. High temperature 25 in either the cytoplasm or periplasm of the cell during pro Pn Alkyl- or halo-benzoates duction. Secretion leaders useful for targeting proteins, e.g., Pl Alkyl- or halo-toluenes hG-CSF, are described elsewhere herein, and in U.S. Pat. Psal Salicylates 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) 30 referenced above. Examples of secretion leaders that can be Manual of Industrial Microbiology and Biotechnology (A. used in the methods of the provided invention are shown in Demain & J. Davies, eds.) pp. 460-74 (ASM Press, Washing Table 3. U.S. Pat. No. 7,070,989 describes a method for ton, D.C.); H. Schweizer (2001) Current Opinion in Biotech secreting G-CSF into the periplasm of an E. coli cell. In some nology, 12:439-445; and R. Slater & R. Williams (2000 embodiments, expression constructs are provided that encode Molecular Biology and Biotechnology (J. Walker & R. Rap 35 hG-CSF fused to a secretion leader that can transporthG-CSF ley, eds.) pp. 125-54 (The Royal Society of Chemistry, Cam to the periplasm of a Pseudomonas cell. In some embodi bridge, UK)). A promoter having the nucleotide sequence of ments, the secretion leader the secretion leader is cleaved a promoter native to the selected bacterial host cell also may from the hC-CSF protein. In some embodiments, the secre be used to control expression of the transgene encoding the tion leader facilitates production of soluble hC-CSF compris target polypeptide, e.g., a Pseudomonas anthranilate or ben 40 ing an N-terminal methionine (Met-G-CSF). Zoate operon promoter (Pant, Pben). Tandem promoters may An expression construct useful in practicing the methods also be used in which more than one promoter is covalently of the present invention can include, in addition to the protein attached to another, whether the same or different in coding sequence, the following regulatory elements operably sequence, e.g., a Pant-Pben tandem promoter (interpromoter linked thereto: a promoter, a ribosome binding site (RBS), a hybrid) or a Plac-Plac tandem promoter, or whether derived 45 transcription terminator, and translational start and stop sig from the same or different organisms. nals. Useful RBSs can be obtained from any of the species Regulated promoters utilize promoter regulatory proteins useful as host cells in expression systems according to, e.g., in order to control transcription of the gene of which the U.S. Pat. App. Pub. No. 2008/0269070 and U.S. patent appli promoter is a part. Where a regulated promoter is used herein, cation Ser. No. 12/610,207. Many specific and a variety of a corresponding promoter regulatory protein will also be part 50 consensus RBSS are known, e.g., those described in and ref of an expression system according to the present invention. erenced by D. Frishman et al., Gene 234(2):257-65 (8 Jul. Examples of promoter regulatory proteins include: activator 1999); and B. E. Suzek et al., Bioinformatics 17(12): 1123-30 proteins, e.g., E. coli catabolite activator protein, MalT pro (December 2001). In addition, either native or synthetic RBSs tein; AraC family transcriptional activators; repressor pro may be used, e.g., those described in: EP 0207459 (synthetic teins, e.g., E. coli Lad proteins; and dual-function regulatory 55 RBSs); O. Ikehata et al., Eur. J. Biochem. 181(3):563-70 proteins, e.g., E. coli NagO protein. Many regulated-pro (1989) (native RBS sequence of AAGGAAG). Further moter/promoter-regulatory-protein pairs are known in the art. examples of methods, vectors, and translation and transcrip In one embodiment, the expression construct for the target tion elements, and other elements useful in the present inven protein(s) and the heterologous protein of interest are under tion are described in, e.g.: U.S. Pat. No. 5,055,294 to Gilroy the control of the same regulatory element. 60 and U.S. Pat. No. 5,128,130 to Gilroy et al.: U.S. Pat. No. Promoter regulatory proteins interact with an effector com 5,281,532 to Rammler et al.; U.S. Pat. Nos. 4,695,455 and pound, i.e., a compound that reversibly or irreversibly asso 4,861,595 to Barnes et al.; U.S. Pat. No. 4,755,465 to Gray et ciates with the regulatory protein so as to enable the protein to al.; and U.S. Pat. No. 5,169,760 to Wilcox. either release or bind to at least one DNA transcription regu Host Strains latory region of the gene that is under the control of the 65 Bacterial hosts, including Pseudomonads, and closely promoter, thereby permitting or blocking the action of a tran related bacterial organisms are contemplated for use in prac Scriptase enzyme in initiating transcription of the gene. Effec ticing the methods of the invention. In certain embodiments, US 8,455,218 B2 11 12 the Pseudomonad host cell is Pseudomonas fluorescens. The TABLE 2-continued host cell can also be an E. coli cell. Host cells and constructs useful in practicing the methods Families and Genera Listed in the Part, “Gram-Negative of the invention can be identified or made using reagents and Aerobic Rods and Cocci' (in Bergey (1974) methods known in the art and described in the literature, e.g., 5 Family III. Rhizobiaceae Agrobacterium Rhizobium in U.S. Pat. App. Pub. No. 2009/0325230, “Protein Expres Family IV. Methylomonadaceae Methylococcus sion Systems, incorporated herein by reference in its Methylomonas entirety. This publication describes production of a recombi Family V. Halobacteriaceae Haiobacterium nant polypeptide by introduction of a nucleic acid construct Haiococcus 10 Other Genera Acetobacter into an auxotrophic Pseudomonas fluorescens host cell com Alcaligenes prising a chromosomal lacI gene insert. The nucleic acid Bordeteia Bruceiia construct comprises a nucleotide sequence encoding the Franciselia recombinant polypeptide operably linked to a promoter Thermits capable of directing expression of the nucleic acid in the host 15 cell, and also comprises a nucleotide sequence encoding an Pseudomonas and closely related bacteria are generally auxotrophic selection marker. The auxotrophic selection part of the group defined as “Gram(-) Proteobacteria Sub marker is a polypeptide that restores prototrophy to the aux group 1 or “Gram-Negative Aerobic Rods and Cocci' otrophic host cell. In embodiments, the cell is auxotrophic for (Buchanan and Gibbons (eds.) (1974) Bergey’s Manual of proline, uracil, or combinations thereof. In embodiments, the Determinative Bacteriology, pp. 217-289). Pseudomonas host cell is derived from MB101 (ATCC deposit PTA-7841). host strains are described in the literature, e.g., in U.S. Pat. U.S. Pat. App. Pub. No. 2009/0325230, “Protein Expression App. Pub. No. 2006/0040352, cited above. Systems.” and in Schneider, et al., 2005, 'Auxotrophic mark “Gram-negative Proteobacteria Subgroup 1 also includes ers pyrF and proC can replace antibiotic markers on protein Proteobacteria that would be classified in this heading production plasmids in high-cell-density Pseudomonas fluo 25 according to the criteria used in the classification. The head rescens fermentation. Biotechnol. Progress 21(2): 343-8. ing also includes groups that were previously classified in this both incorporated herein by reference in their entirety, section but are no longer, Such as the genera Acidovorax, describe a production host strain auxotrophic for uracil that Brevundimonas, Burkholderia, Hydrogenophaga, Oceani was constructed by deleting the pyrF gene in strain MB 101. monas, Ralstonia, and Stenotrophomonas, the genus Sphin The pyrE gene was cloned from strain MB214 (ATCC deposit 30 gomonas (and the genus Blastomonas, derived therefrom), PTA-7840) to generate a plasmid that can complement the which was created by regrouping organisms belonging to pyrF deletion to restore prototrophy. In particular embodi (and previously called species of) the genus Xanthomonas, ments, a dual pyrF-proC dual auxotrophic selection marker the genus Acidomonas, which was created by regrouping system in a Pfluorescens host cell is used. A PyrE production organisms belonging to the genus Acetobacter as defined in host strain as described can be used as the background for 35 Bergey (1974). In addition hosts can include cells from the introducing other desired genomic changes, including those genus Pseudomonas, Pseudomonas enalia (ATCC 14393), described herein as useful in practicing the methods of the Pseudomonas nigrifaciensi (ATCC 19375), and Pseudomo invention. nas putrefaciens (ATCC 8071), which have been reclassified In embodiments, the host cell is of the order Pseudomonad respectively as Alteromonas haloplanktis, Alteromonas nigri ales. Where the host cell is of the order Pseudomonadales, it 40 faciens, and Alteromonas putrefaciens. Similarly, e.g., may be a member of the family Pseudomonadaceae, includ Pseudomonas acidovorans (ATCC 15668) and Pseudomonas ing the genus Pseudomonas. Gamma Proteobacterial hosts testosteroni (ATCC 1 1996) have since been reclassified as include members of the species Escherichia coli and mem Comamonas acidovorans and Comamonas testosteroni, bers of the species Pseudomonas fluorescens. respectively; and Pseudomonas nigrifaciens (ATCC 19375) Other Pseudomonas organisms may also be useful. 45 and Pseudomonas piscicida (ATCC 15057) have been reclas Pseudomonads and closely related species include Gram sified respectively as Pseudoalteromonas nigrifaciens and negative Proteobacteria Subgroup 1, which include the group Pseudoalteromonas piscicida. “Gram-negative Proteobacte of Proteobacteria belonging to the families and/or genera ria Subgroup 1 also includes Proteobacteria classified as described as “Gram-Negative Aerobic Rods and Cocci' by R. belonging to any of the families: Pseudomonadaceae, AZoto E. Buchanan and N. E. Gibbons (eds.), Bergey’s Manual of bacteraceae (now often called by the synonym, the Azoto Determinative Bacteriology, pp. 217-289 (8th ed., 1974) (The bacter group' of Pseudomonadaceae), Rhizobiaceae, and Williams & Wilkins Co., Baltimore, Md., USA) (hereinafter Methylomonadaceae (now often called by the synonym, “Bergey (1974)). Table 2 presents these families and genera “Methylococcaceae). Consequently, in addition to those of organisms. genera otherwise described herein, further Proteobacterial 55 genera falling within “Gram-negative Proteobacteria Sub group 1 include: 1) Azotobacter group bacteria of the genus TABLE 2 Azorhizophilus; 2) Pseudomonadaceae family bacteria of the Families and Genera Listed in the Part, “Gram-Negative genera Cellvibrio, Oligella, and Teredinibacter; 3) Rhizobi Aerobic Rods and Cocci' (in Bergey (1974) aceae family bacteria of the genera Chelatobacter, Ensifer, Family I. Pseudomonaceae Giuconobacter 60 Liberibacter (also called “Candidatus Liberibacter'), and Pseudomonas Sinorhizobium; and 4) Methylococcaceae family bacteria of Xanthomonas the genera Methylobacter, Methylocaldum, Methylomicro Zoogloea bium, Methylosarcina, and Methylosphaera. Family II. Azotobacteraceae Azomonas Azotobacter The host cell can be selected from “Gram-negative Proteo Beijerinckia 65 bacteria Subgroup 16.” “Gram-negative Proteobacteria Sub Dexia group 16' is defined as the group of Proteobacteria of the following Pseudomonas species (with the ATCC or other US 8,455,218 B2 13 14 deposit numbers of exemplary strain(s) shown in parenthe ria Subgroup 17 is defined as the group of Proteobacteria sis): Pseudomonas abietaniphila (ATCC 700689); known in the art as the “fluorescent Pseudomonads' includ Pseudomonas aeruginosa (ATCC 101.45); Pseudomonas ing those belonging, e.g., to the following Pseudomonas spe alcaligenes (ATCC 14909); Pseudomonas anguilliseptica cies: Pseudomonas azotoformans, Pseudomonas brenneri; (ATCC 33660); Pseudomonas citronellolis (ATCC 13674): Pseudomonas cedrella, Pseudomonas COrrugata, Pseudomonas flavescens (ATCC 51555); Pseudomonas men Pseudomonas extremorientalis, Pseudomonas fluorescens, docina (ATCC 25411); Pseudomonas nitroreducens (ATCC Pseudomonas gessardii. Pseudomonas libanensis, 33634); Pseudomonas oleovorans (ATCC 8062); Pseudomo Pseudomonas mandelii, Pseudomonas marginalis, nas pseudoalcaligenes (ATCC 17440); Pseudomonas resino Pseudomonas migulae, Pseudomonas mucidolens, vorans (ATCC 14235); Pseudomonas straminea (ATCC 10 Pseudomonas Orientalis, Pseudomonas rhodesiae, 33.636); Pseudomonas agarici (ATCC 25941); Pseudomonas Pseudomonas synxantha, Pseudomonas tolaasii; and alcaliphila, Pseudomonas alginovora, Pseudomonas ander Pseudomonas veronii. sonii. Pseudomonas asplenii (ATCC 23835); Pseudomonas Proteases azelaica (ATCC 27162): Pseudomonas beverinckii (ATCC In one embodiment, the methods of the provided invention 19372); Pseudomonas borealis, Pseudomonas boreopolis 15 comprise using a Pseudomonas host cell, comprising one or (ATCC 33662); Pseudomonas brassicacearum, Pseudomo more mutations (e.g., a partial or complete deletion) in one or nas butanovora (ATCC 43655); Pseudomonas cellulosa more protease genes, to produce recombinant hC-CSF pro (ATCC 55703); Pseudomonas aurantiaca (ATCC 33663); tein. In some embodiments, a mutation in a protease gene can Pseudomonas chlororaphis (ATCC 9446, ATCC 13985, facilitate generation of recombinant hC-CSF protein com ATCC 17418, ATCC 17461); Pseudomonas fragi (ATCC prising an intact methionine at the N-terminus (Met-G-CSF). 4973); Pseudomonas lundensis (ATCC 49968); Pseudomo Exemplary target protease genes include those proteases nas taetrolens (ATCC 4683); Pseudomonascissicola (ATCC classified as Aminopeptidases; Dipeptidases; Dipeptidyl 33616); Pseudomonas coronafaciens, Pseudomonas diter peptidases and tripeptidyl peptidases; Peptidyl-dipeptidases; peniphila, Pseudomonas elongata (ATCC 10144); Serine-type carboxypeptidases; Metallocarboxypeptidases; Pseudomonasflectens (ATCC 12775); Pseudomonas azoto 25 Cysteine-type carboxypeptidases; Omegapeptidases; Serine formans, Pseudomonas brenneri. Pseudomonas cedrella, proteinases; Cysteine proteinases; Aspartic proteinases; Met Pseudomonas corrugata (ATCC 29736); Pseudomonas allo proteinases; or Proteinases of unknown mechanism. extremorientalis, Pseudomonas fluorescens (ATCC 35858); Aminopeptidases include cytosol aminopeptidase (leucyl Pseudomonas gessardii. Pseudomonas libanensis, aminopeptidase), membrane alanyl aminopeptidase, cystinyl Pseudomonas mandelii (ATCC 700871); Pseudomonas mar 30 aminopeptidase, tripeptide aminopeptidase, prolyl ami ginalis (ATCC 10844); Pseudomonas migulae, Pseudomo nopeptidase, arginyl aminopeptidase, glutamyl aminopepti nas mucidolens (ATCC 4685); Pseudomonas orientalis, dase, X-pro aminopeptidase, bacterial leucylaminopeptidase, Pseudomonas rhodesiae, Pseudomonas synxantha (ATCC thermophilic aminopeptidase, clostridial aminopeptidase, 9890); Pseudomonas tolaasii (ATCC 33618); Pseudomonas cytosol alanyl aminopeptidase, lysyl aminopeptidase, X-trp veronii (ATCC 700474); Pseudomonas federiksbergensis, 35 aminopeptidase, tryptophanyl aminopeptidase, methionyl Pseudomonas geniculata (ATCC 19374); Pseudomonas gin aminopeptidas, d-stereospecific aminopeptidase, aminopep geri. Pseudomonas graminis, Pseudomonas grimontii, tidase ey. Dipeptidases include X-his dipeptidase, X-arg Pseudomonas halodenitrificans, Pseudomonas halophila, dipeptidase, X-methyl-his dipeptidase, cys-gly dipeptidase, Pseudomonas hibiscicola (ATCC 19867); Pseudomonas hut glu-glu dipeptidase, pro-X dipeptidase, X-pro dipeptidase, tiensis (ATCC 14670); Pseudomonas hydrogenovora, 40 met-X dipeptidase, non-stereospecific dipeptidase, cytosol Pseudomonas jessenii (ATCC 700870); Pseudomonas kilon non-specific dipeptidase, membrane dipeptidase, beta-ala-his ensis, Pseudomonas lanceolata (ATCC 14669); Pseudomo dipeptidase. Dipeptidyl-peptidases and tripeptidyl peptidases nas lini. Pseudomonas marginate (ATCC 25417); include dipeptidyl-peptidasei, dipeptidyl-peptidase ii, dipep Pseudomonas mephitica (ATCC 33665); Pseudomonas deni tidyl peptidase iii, dipeptidyl-peptidase iv, dipeptidyl-dipep trificans (ATCC 19244); Pseudomonas pertucinogena 45 tidase, tripeptidyl-peptidase I, tripeptidyl-peptidase II. Pepti (ATCC 190); Pseudomonas pictorum (ATCC 23328); dyl-dipeptidases include peptidyl-dipeptidase a and peptidyl Pseudomonas psychrophila, Pseudomonas filva (ATCC dipeptidase b. Serine-type carboxypeptidases include 31418); Pseudomonas monteilii (ATCC 700476); Pseudomo lysosomal pro-X carboxypeptidase, serine-type D-ala-D-ala nas mosselii, Pseudomonas oryzihabitans (ATCC 43272): carboxypeptidase, carboxypeptidase C, carboxypeptidase D. Pseudomonas plecoglossicida (ATCC 700383); Pseudomo 50 Metallocarboxypeptidases include carboxypeptidase a, car nas putida (ATCC 12633); Pseudomonas reactans, boxypeptidase B, lysine(arginine) carboxypeptidase, gly-X Pseudomonas spinosa (ATCC 14606); Pseudomonas bale carboxypeptidase, alanine carboxypeptidase, muramoylpen arica, Pseudomonas luteola (ATCC 43273); Pseudomonas tapeptide carboxypeptidase, carboxypeptidase h, glutamate Stutzeri (ATCC 17588); Pseudomonas amygdali (ATCC carboxypeptidase, carboxypeptidase M. muramoyltetrapep 33614); Pseudomonas avellanae (ATCC 700331); 55 tide carboxypeptidase, Zinc d-ala-d-ala carboxypeptidase, Pseudomonas caricapapayae (ATCC 33615); Pseudomonas carboxypeptidase A2, membrane pro-X carboxypeptidase, cichorii (ATCC 10857); Pseudomonas ficuserectae (ATCC tubulinyl-tyr carboxypeptidase, carboxypeptidase t. Omega 35104); Pseudomonas fiscovaginae, Pseudomonas meliae peptidases include acylaminoacyl-peptidase, peptidyl-glyci (ATCC 33050); Pseudomonas syringae (ATCC 19310): namidase, pyroglutamyl-peptidase I, beta-aspartyl-pepti Pseudomonas viridiflava (ATCC 13223); Pseudomonas ther 60 dase, pyroglutamyl-peptidase II, n-formylmethionyl mocarboxydovorans (ATCC 35961); Pseudomonas thermo peptidase, pteroylpoly-gamma-glutamate tolerans, Pseudomonas thivervalensis, Pseudomonas van carboxypeptidase, gamma-glu-X carboxypeptidase, acylmu couverensis (ATCC 700688); Pseudomonas wisconsinensis; ramoyl-ala peptidase. Serine proteinases include chymot and Pseudomonas xiamenensis. In one embodiment, the host rypsin, chymotrypsin c, metridin, trypsin, thrombin, coagul cell for expression of hC-CSF is Pseudomonas fluorescens. 65 lation factor Xa, plasmin, enteropeptidase, acrosin, alpha The host cell can also be selected from “Gram-negative lytic protease, glutamyl, endopeptidase, cathepsin G, Proteobacteria Subgroup 17.” “Gram-negative Proteobacte coagulation factor Viia, coagulation factorixa, cucumisi, pro US 8,455,218 B2 15 16 lyl oligopeptidase, coagulation factor Xia, brachyurin, plasma (methionine aminopeptidase); and Serine Peptidase S26 sig kallikrein, tissue kallikrein, pancreatic elastase, leukocyte nal peptidase I family member RXFO 1181.1 (signal pepti elastase, coagulation factor Xia, chymase, complement com dase). ponent c1rS5, complement component c1 S55, classical Codon Optimization complement pathway c3/c5 convertase, complement factor I. In one embodiment, the methods of the provided invention complement factor D, alternative-complement pathway c3/c5 comprise expression of recombinant hC-CSF from a con convertase, cerevisin, hypodermin C, lysyl endopeptidase, struct that has been optimized for codon usage in a strain of endopeptidase 1a, gamma-reni, Venombin ab, leucyl interest. In embodiments, the strain is a Pseudomonas host endopeptidase, tryptase, Scutelarin, kexin, Subtilisin, oryzin, cell, e.g., Pseudomonas fluorescens. Methods for optimizing endopeptidase k, thermomycolin, thermitase, endopeptidase 10 SO, T-plasminogen activator, protein C, pancreatic endopep codons to improve expression in bacterial hosts are known in tidase E, pancreatic elastase ii, IGA-specific serine endopep the art and described in the literature. For example, optimi tidase, U-plasminogen, activator, Venombin A, furin, myelo Zation of codons for expression in a Pseudomonas host strain blastin, semenogelase, granzyme A or cytotoxic is described, e.g., in U.S. Pat. App. Pub. No. 2007/0292918, T-lymphocyte proteinase 1, granzyme B or cytotoxic T-lym 15 “Codon Optimization Method, incorporated herein by ref phocyte proteinase 2, Streptogrisin A, treptogrisin B, erence in its entirety. glutamylendopeptidase II, oligopeptidase B, limulus clotting In heterologous expression systems, optimization steps factor c, limulus clotting factor, limulus clotting enzyme, may improve the ability of the host to produce the foreign omptin, repressor lexa, bacterial leader peptidase I, togavirin, protein. Protein expression is governed by a host of factors flavirin. Cysteine proteinases include cathepsin B, papain, including those that affect transcription, mRNA processing, ficin, chymopapain, asclepain, clostripain, Streptopain, and stability and initiation of translation. The polynucleotide actinide, cathepsin 1, cathepsin H, calpain, cathepsint, gly optimization steps may include steps to improve the ability of cyl, endopeptidase, cancer procoagulant, cathepsin S, picor the host to produce the foreign protein as well as steps to assist nain 3C, picornain 2A, caricain, ananain, stem bromelain, the researcher in efficiently designing expression constructs. fruit bromelain, legumain, histolysain, interleukin 1-beta 25 Optimization strategies may include, for example, the modi converting enzyme. Aspartic proteinases include pepsin A, fication of translation initiation regions, alteration of mRNA pepsin B, gastricsin, chymosin, cathepsin D. neopenthesin, structural elements, and the use of different codon biases. renin, retropepsin, pro-opiomelanocortin converting enzyme, Methods for optimizing the nucleic acid sequence of to aspergillopepsin I, aspergillopepsin II, penicillopepsin, improve expression of a heterologous protein in a bacterial rhizopuspepsin, endothiapepsin, mucoropepsin, candidapep 30 host are known in the art and described in the literature. For sin, saccharopepsin, rhodotorulapepsin, physaropepsin, acro example, optimization of codons for expression in a cylindropepsin, polyporopepsin, pycnoporopepsin, Scytali Pseudomonas host strain is described, e.g., in U.S. Pat. App. dopepsina, Scytalidopepsin b, Xanthomonapepsin, cathepsin Pub. No. 2007/0292918, “Codon Optimization Method.” e, barrierpepsin, bacterial leader peptidase I. incorporated herein by reference in its entirety. pseudomonapepsin, plasmepsin. Metallo proteinases include 35 Optimization canthus address any of a number of sequence atrolysina, microbial collagenase, leucolysin, interstitial col features of the heterologous gene. As a specific example, a lagenase, neprilysin, envelysin, iga-specific metalloendopep rare codon-induced translational pause can result in reduced tidase, procollagen N-endopeptidase, thimet oligopeptidase, heterologous protein expression. A rare codon-induced trans neurolysin, stromelysin 1, meprinA, procollagen C-endopep lational pause includes the presence of codons in the poly tidase, peptidyl-lys metalloendopeptidase, astacin, stromel 40 nucleotide of interest that are rarely used in the host organism ysin, 2., matrilysin gelatinase, aeromonolysin, pseudo lysin, may have a negative effect on protein translation due to their thermolysin, bacillolysin, aureolysin, coccolysin, mycolysin, scarcity in the available tRNA pool. One method of improv beta-lytic metalloendopeptidase, peptidyl-asp metalloen ing optimal translation in the host organism includes per dopeptidase, neutrophil collagenase, gelatinase B, leish forming codon optimization which can result in rare host manolysin, saccharolysin, autolysin, deuterolysin, Serralysin, 45 codons being removed from the synthetic polynucleotide atrolysin B, atrolysin C. atroxase, atrolysin E. atrolysin F. Sequence. adamalysin, horrilysin, ruberlysin, bothropasin, bothrolysin, Alternate translational initiation also can result in reduced ophiolysin, trimerelysin I, trimerelysin II, mucrolysin, pitril heterologous protein expression. Alternate translational ini ysin, insulysin, O-Syaloglycoprotein endopeptidase, russell tiation can include a synthetic polynucleotide sequence inad ysin, mitochondrial, intermediate, peptidase, dactylysin, nar 50 Vertently containing motifs capable of functioning as a ribo dilysin, magnolysin, meprin B, mitochondrial processing some binding site (RBS). These sites can result in initiating peptidase, macrophage elastase, choriolysin, toxilysin. Pro translation of a truncated protein from a gene-internal site. teinases of unknown mechanism include thermopsin and One method of reducing the possibility of producing a trun multicatalytic endopeptidase complex. cated protein, which can be difficult to remove during purifi Certain proteases can have both protease and chaperone 55 cation, includes eliminating putative internal RBS sequences like activity. When these proteases are negatively affecting from an optimized polynucleotide sequence. protein yield and/or quality it can be useful to specifically Repeat-induced polymerase slippage can result in reduced delete their protease activity, and they can be overexpressed heterologous protein expression. Repeat-induced polymerase when their chaperone activity may positively affect protein slippage involves nucleotide sequence repeats that have been yield and/or quality. These proteases include, but are not 60 shown to cause slippage or Stuttering of DNA polymerase limited to: Hsp100(Clp/Hsl) family members RXF04587.1 which can result in frameshift mutations. Such repeats can (clpA), RXF08347.1, RXF04654.2 (clpX), RXF04663.1, also cause slippage of RNA polymerase. In an organism with RXFO1957.2 (hslU), RXFO 1961.2 (hslV); Peptidyl-prolyl a high G+C content bias, there can be a higher degree of cis-trans isomerase family member RXF05345.2 (ppiB); repeats composed of G or C nucleotide repeats. Therefore, Metallopeptidase M20 family member RXF04892.1 (amino 65 one method of reducing the possibility of inducing RNA hydrolase); Metallopeptidase M24 family members polymerase slippage, includes altering extended repeats of G RXF04693.1 (methionine aminopeptidase) and RXF03364.1 or C nucleotides. US 8,455,218 B2 17 18 Interfering secondary structures also can result in reduced Pseudomonas host cell, codons occurring at less than 10% in heterologous protein expression. Secondary structures can the Pseudomonas host cell, a rare codon-induced transla sequester the RBS sequence or initiation codon and have been tional pause, a putative internal RBS sequence, an extended correlated to a reduction in protein expression. Stemloop repeat of G or C nucleotides, an interfering secondary struc structures can also be involved in transcriptional pausing and ture, a restriction site, or combinations thereof. attenuation. An optimized polynucleotide sequence can con Furthermore, the amino acid sequence of any secretion tain minimal secondary structures in the RBS and gene cod leader useful in practicing the methods of the present inven ing regions of the nucleotide sequence to allow for improved tion can be encoded by any appropriate nucleic acid transcription and translation. sequence. Codon optimization for expression in E. coli is Another feature that can effect heterologous protein 10 described, e.g., by Welch, et al., 2009, PLoS One, “Design expression is the presence of restriction sites. By removing Parameters to Control Synthetic Gene Expression in Escheri restriction sites that could interfere with subsequent sub chia coli. 4(9): e7002, Ghane, et al., 2008, Krishna R. et al., cloning of transcription units into host expression vectors a (2008) Mol Biotechnology "Optimization of the AT-content polynucleotide sequence can be optimized. of Codons Immediately Downstream of the Initiation Codon For example, the optimization process can begin by iden 15 and Evaluation of Culture Conditions for High-level Expres tifying the desired amino acid sequence to be heterologously sion of Recombinant Human G-CSF in Escherichia coli.” expressed by the host. From the amino acid sequence a can 38:221-232. didate polynucleotide or DNA sequence can be designed. High Throughput Screens During the design of the synthetic DNA sequence, the fre In some embodiments, a high throughput Screen can be quency of codon usage can be compared to the codon usage of conducted to determine optimal conditions for expressing the host expression organism and rare host codons can be soluble recombinant hCG-CSF. In some embodiments, a high removed from the synthetic sequence. Additionally, the Syn throughput screen can be conducted to determine optimal thetic candidate DNA sequence can be modified in order to conditions for expressing soluble recombinant hCG-CSF com remove undesirable enzyme restriction sites and add or prising an N-terminal methionine (Met-G-CSF). The condi remove any desired signal sequences, linkers or untranslated 25 tions that be varied in the screen include, for example, the host regions. The synthetic DNA sequence can be analyzed for the cell, genetic background of the host cell (e.g., deletions of presence of secondary structure that may interfere with the different proteases), type of promoter in an expression con translation process, such as G/C repeats and stem-loop struc struct, type of secretion leader fused to encoded hC-CSF, tures. Before the candidate DNA sequence is synthesized, the temperature of growth, OD of induction when an inducible optimized sequence design can be checked to Verify that the 30 promoter is used, amount of IPTG used for induction when a sequence correctly encodes the desired amino acid sequence. lacZ promoteris used, duration of protein induction, tempera Finally, the candidate DNA sequence can be synthesized ture of growth following addition of an inducing agent to a using DNA synthesis techniques, such as those known in the culture, rate of agitation of culture, method of selection for art. plasmid maintenance, Volume of culture in a vessel, and In another embodiment of the invention, the general codon 35 method of cell lysing. usage in a host organism, such as P. fluorescens, can be In some embodiments, a library (or “array') of host strains utilized to optimize the expression of the heterologous poly is provided, wherein each strain (or “population of host nucleotide sequence. The percentage and distribution of cells') in the library has been genetically modified to modu codons that rarely would be considered as preferred for a late the expression of one or more target genes in the host cell. particular amino acid in the host expression system can be 40 An “optimal host strain” or “optimal expression system’ can evaluated. Values of 5% and 10% usage can be used as cutoff be identified or selected based on the quantity, quality, and/or values for the determination of rare codons. For example, the location of the expressed protein of interest compared to other codons listed in Table 3 have a calculated occurrence of less populations of phenotypically distinct host cells in the array. than 5% in the Pfluorescens MB214 genome and would be Thus, an optimal host strain is the strain that produces the generally avoided in an optimized gene expressed in a P 45 polypeptide of interest according to a desired specification. While the desired specification will vary depending on the fluorescens host. polypeptide being produced, the specification includes the quality and/or quantity of protein, whether the protein is TABLE 3 sequestered or secreted, protein folding, and the like. For Codons occurring at less than 5% in Pfluorescens MB214 50 example, the optimal host strain or optimal expression system produces a yield, characterized by the amount of soluble Amino Acid (s) Codon(s) Used % Occurrence heterologous protein, the amount of recoverable heterologous G Gly GGA 3.26 protein, the amount of properly processed heterologous pro IIle ATA 3.05 tein, the amount of properly folded heterologous protein, the L. Leu CTA 1.78 55 amount of active heterologous protein, and/or the total CTT 4.57 TTA 1.89 amount of heterologous protein, of a certain absolute level or R Arg AGA 1.39 a certain level relative to that produced by an indicator strain, AGG 2.72 i.e., a strain used for comparison. CGA 4.99 Methods of screening microbial hosts to identify strains S Ser TCT 4.28 60 with improved yield and/or quality in the expression of het erologous proteins are described, for example, in U.S. Patent The present invention contemplates the use of any GCSF Application Publication No. 20080269070. coding sequence, including any sequence that has been opti Bacterial Growth Conditions mized for expression in the Pseudomonas host cell being Growth conditions useful in the methods of the provided used. Sequences contemplated for use can be optimized to 65 invention can comprise a temperature of about 4°C. to about any degree as desired, including, but not limited to, optimi 42°C. and a pH of about 5.7 to about 8.8. When an expression Zation to eliminate: codons occurring at less than 5% in the construct with a lacz promoter is used, expression can be US 8,455,218 B2 19 20 induced by adding IPTG to a culture at a final concentration of In other embodiments, the OD575 is about 80 to about 100, about 0.01 mM to about 1.0 mM. about 100 to about 120, about 120 to about 140, about 140 to The pH of the culture can be maintained using pH buffers about 160. In other embodiments, the OD575 is about 80 to and methods known to those of skill in the art. Control of pH about 120, about 100 to about 140, or about 120 to about 160. during culturing also can be achieved using aqueous ammo In other embodiments, the OD575 is about 80 to about 140, or nia. In embodiments, the pH of the culture is about 5.7 to about 100 to 160. The cell density can be measured by other about 8.8. In certain embodiments, the pH is about 5.7. 5.8, methods and expressed in other units, e.g., in cells per unit 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, volume. For example, an OD575 of about 80 to about 160 of 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, a Pseudomonas fluorescens culture is equivalent to approxi 8.7, or 8.8 In other embodiments, the pH is about 5.7 to 5.9, 10 mately 8x10" to about 1.6x10' colony forming units permL 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, or 35 to 70 g/L dry cell weight. In embodiments, the cell 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, density at the time of culture induction is equivalent to the cell 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, density as specified herein by the absorbance at OD575, 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, regardless of the method used for determining cell density or 8.3 to 8.6, 8.4 to 8.7, or 8.5 to 8.8. In yet other embodiments, 15 the units of measurement. One of skill in the art will know 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 how to make the appropriate conversion for any cell culture. to 6.4, or 6.2 to 6.5. In certain embodiments, the pH is about In embodiments, the final IPTG concentration of the cul 5.7 to about 6.25. ture is about 0.01 mM, about 0.02 mM, about 0.03 mM, about In embodiments, the growth temperature is maintained at 0.04 mM, about 0.05 mM, about 0.06 mM, about 0.07 mM, about 4° C. to about 42° C. In certain embodiments, the about 0.08 mM, about 0.09 mM, about 0.1 mM, about 0.2 growth temperature is about 4°C., about 5°C., about 6°C., mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 about 7°C., about 8°C., about 9°C., about 10°C., about 11° mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, or about 1 C., about 12° C., about 13°C., about 14° C., about 15° C., mM. In other embodiments, the final IPTG concentration of about 16°C., about 17°C., about 18°C., about 19°C., about the culture is about 0.08 mM to about 0.1 mM, about 0.1 mM 20°C., about 21°C., about 22°C., about 23°C., about 24°C., 25 to about 0.2 mM, about 0.2 mM to about 0.3 mM, about 0.3 about 25°C., about 26°C., about 27°C., about 28°C., about mM to about 0.4 mM, about 0.2 mM to about 0.4 mM, about 29°C., about 30°C., about 31°C., about 32°C., about 33°C., 0.08 to about 0.2 mM, or about 0.1 to 1 mM. about 34°C., about 35°C., about 36°C., about 37°C., about In embodiments wherein a non-lac type promoter is used, 38°C., about 39°C., about 40°C., about 41° C., or about 42° as described herein and in the literature, other inducers or C. In other embodiments, the growth temperature is main 30 effectors can be used. In one embodiment, the promoter is a tained at about 25°C. to about 27°C., about 25°C. to about constitutive promoter. 28°C., about 25°C. to about 29° C., about 25°C. to about 30° After adding and inducing agent, cultures can be grown for C., about 25°C. to about 31°C., about 25°C. to about 32°C., a period of time, for example about 24 hours, during which about 25°C. to about 33°C., about 26° C. to about 28°C., time the recombinant hCG-CSF is expressed. After adding an about 26°C. to about 29° C., about 26° C. to about 30° C., 35 inducing agent, a culture can be grown for about 1 hr, about 2 about 26°C. to about 31° C., about 26° C. to about 32° C., hr, about 3 hr, about 4 hr, about 5 hr, about 6 hr, about 7 hr, about 27°C. to about 29° C., about 27°C. to about 30° C., about 8 hr, about 9 hr, about 10 hr, about 11 hr., about 12 hr. about 27°C. to about 31° C., about 27°C. to about 32° C., about 13 hr, about 14 hr, about 15 hr, about 16 hr, about 17 hr, about 26°C. to about 33°C., about 28°C. to about 30° C., about 18 hr, about 19 hr, about 20 hr, about 21 hr., about 22 hr. about 28°C. to about 31° C., about 28°C. to about 32° C., 40 about 23 hr, about 24 hr, about 36 hr, or about 48 hr. After an about 29° C. to about 31° C., about 29° C. to about 32° C., inducing agent is added to a culture, the culture can be grown about 29° C. to about 33°C., about 30° C. to about 32° C., for about 1 to 48 hrs, about 1 to 24 hrs, about 10 to 24 hrs, about 30° C. to about 33°C., about 31° C. to about 33°C., about 15 to 24 hrs, or about 20 to 24 hrs. Cell cultures can be about 31° C. to about 32°C., about 30° C. to about 33°C., or concentrated by centrifugation, and the culture pellet resus about 32° C. to about 33° C. In other embodiments, the 45 pended in a buffer or solution appropriate for the Subsequent temperature is changed during culturing. In one embodiment, lysis procedure. the temperature is maintained at about 30°C. before an agent In embodiments, cells are disrupted using equipment for to induce expression from the construct expressing hC-CSF is high pressure mechanical cell disruption (which are available added to the culture, and the temperature is dropped to about commercially, e.g., Microfluidics Microfluidizer, Constant 25°C. after adding an agent to induce expression, e.g., IPTG 50 Cell Disruptor, Niro-Soavi homogenizer or APV-Gaulin is added to the culture. homogenizer). Cells expressing hC-CSF can be disrupted, for Induction example, using Sonication. Any appropriate method known in As described elsewhere herein, inducible promoters can be the art for lysing cells can be used to release the soluble used in the expression construct to control expression of the fraction. For example, in embodiments, chemical and/or recombinant hG-CSF, e.g., a lac promoter. In the case of the 55 enzymatic cell lysis reagents, such as cell-wall lytic enzyme lac promoter derivatives or family members, e.g., the tac and EDTA, can be used. Use of frozen or previously stored promoter, the effector compound is an inducer, such as a cultures is also contemplated in the methods of the invention. gratuitous inducer like IPTG (isopropyl-3-D-1-thiogalacto Cultures can be OD-normalized prior to lysis. For example, pyranoside, also called "isopropylthiogalactoside'). In cells can be normalized to an OD600 of about 10, about 11, embodiments, a lac promoter derivative is used, and hC-CSF 60 about 12, about 13, about 14, about 15, about 16, about 17, expression is induced by the addition of IPTG to a final about 18, about 19, or about 20. concentration of about 0.01 mM to about 1.0 mM, when the Centrifugation can be performed using any appropriate cell density has reached a level identified by an OD575 of equipment and method. Centrifugation of cell culture or about 80 to about 160. In embodiments, the OD575 at the time lysate for the purposes of separating a soluble fraction from of culture induction for hC-CSF can be about 80, about 90, 65 an insoluble fraction is well-known in the art. For example, about 100, about 110, about 120, about 130, about 140, about lysed cells can be centrifuged at 20,800xg for 20 minutes (at 150, about 160, about 170 about 180. 4°C.), and the Supernatants removed using manual or auto US 8,455,218 B2 21 22 mated liquid handling. The pellet (insoluble) fraction is resus liters to about 25 liters, about 25 liters to about 50 liters, or pended in a buffered solution, e.g., phosphate buffered saline about 50 liters to about 100 liters. In other embodiments, the (PBS), pH 7.4. Resuspension can be carried out using, e.g., fermentation volume is at or above 5 Liters, 10 Liters, 15 equipment Such as impellers connected to an overhead mixer, Liters, 20 Liters, 25 Liters, 50 Liters, 75 Liters, 100 Liters, magnetic stir-bars, rocking shakers, etc. 5 200 Liters, 500 Liters, 1,000 Liters, 2,000 Liters, 5,000 A “soluble fraction, i.e., the soluble supernatant obtained Liters, 10,000 Liters, or 50,000 Liters. after centrifugation of a lysate, and an “insoluble fraction.” Protein Analysis i.e., the pellet obtained after centrifugation of a lysate, result In embodiments, recombinant hCG-CSF protein (e.g., Met from lysing and centrifuging the cultures. These two fractions G-CSF) produced by the methods of the provided invention is also can be referred to as a “first soluble fraction' and a “first 10 analyzed. Recombinant hCG-CSF protein (e.g., Met-G-CSF) insoluble fraction.” respectively. US Patent Application Pub can be analyzed, for example, by biolayer interferometry, lication No. 20050283000 describes means for manipulating SDS-PAGE, Western blot, Far Western blot, ELISA, absor G-CSF protein expressed in inclusion bodies from E. coli. bance, or mass spectrometry (e.g., tandem mass spectrom Fermentation Format etry). The recombinant hC-CSF protein can comprise an In one embodiment, fermentation is used in the methods of 15 N-terminal methionine. producing recombinant hC-CSF comprising an N-terminal In some embodiments, the concentration and/or amounts methionine. The expression system according to the present of recombinanthC-CSF protein (e.g., Met-G-CSF) generated invention can be cultured in any fermentation format. For are determined, for example, by Bradford assay, absorbance, example, batch, fed-batch, semi-continuous, and continuous Coosmassie staining, mass spectrometry, etc. fermentation modes may be employed herein. Protein yield in the insoluble and soluble fractions as In embodiments, the fermentation medium may be described herein can be determined by methods known to selected from among rich media, minimal media, and mineral those of skill in the art, for example, by capillary gel electro salts media. In other embodiments either a minimal medium phoresis (CGE), and Western blot analysis. Soluble fractions or a mineral salts medium is selected. In certain embodi can be evaluated, for example, using biolayer interferometry. ments, a mineral salts medium is selected. 25 Useful measures of protein yield include, e.g., the amount Mineral salts media consists of mineral salts and a carbon of recombinant protein per culture Volume (e.g., grams or Source Such as, e.g., glucose. Sucrose, or glycerol. Examples milligrams of protein/liter of culture), percent or fraction of of mineral salts media include, e.g., M9 medium, Pseudomo recombinant protein measured in the insoluble pellet nas medium (ATCC 179), and Davis and Mingioli medium obtained after lysis (e.g., amount of recombinant protein in (see, BD Davis & ESMingioli (1950).J. Bact. 60:17-28). The 30 extract Supernatant/amount of protein in insoluble fraction), mineral salts used to make mineral salts media include those percent or fraction of active protein (e.g., amount of active Selected from among, e.g., potassium phosphates, ammo proteinfamount protein used in the assay), percent or fraction nium Sulfate or chloride, magnesium Sulfate or chloride, and of total cell protein (tcp), amount of protein/cell, and percent trace minerals such as calcium chloride, borate, and Sulfates dry biomass. of iron, copper, manganese, and Zinc. Typically, no organic 35 In embodiments, the methods of the present invention can nitrogen source. Such as peptone, tryptone, amino acids, or a be used to obtain a yield of soluble recombinant hC-CSF yeast extract, is included in a mineral salts medium. Instead, protein comprising an N-terminal methionine (Met-G-CSF) an inorganic nitrogen source is used and this may be selected of about 0.1 grams per liter to about 10 grams per liter. In from among, e.g., ammonium salts, aqueous ammonia, and certain embodiments, the yield of soluble recombinant hC gaseous ammonia. A mineral salts medium will typically 40 CSF protein comprising an N-terminal methionine (Met-G- contain glucose or glycerol as the carbon Source. In compari CSF) is about 0.1 grams per liter, about 0.2 grams per liter, son to mineral salts media, minimal media can also contain about 0.3 grams per liter, about 0.4 grams per liter, about 0.5 mineral salts and a carbon Source, but can be supplemented grams per liter, about 0.6 grams per liter, about 0.7 grams per with, e.g., low levels of amino acids, vitamins, peptones, or liter, about 0.8 grams per liter, about 0.9 grams per liter, about other ingredients, though these are added at very minimal 45 1 gram per liter, about 2 grams per liter, about 3 grams per levels. Media can be prepared using the methods described in liter, about 4 grams per liter, about 5 grams per liter, about 6 the art, e.g., in U.S. Pat. App. Pub. No. 2006/0040352, refer grams per liter, about 7 grams per liter, about 8 grams per liter, enced and incorporated by reference above. Details of culti about 9 grams per liter, about 10 grams per liter, about 0.1 Vation procedures and mineral salts media useful in the meth grams per liter to about 0.5 grams per liter, about 0.1 grams to ods of the present invention are described by Riesenberg, Det 50 about 1 grams per liter, about 0.1 gram per liter to about 2 al., 1991, “High cell density cultivation of Escherichia coli at grams per liter, about 0.1 grams per liter to about 3 grams per controlled specific growth rate.” J. Biotechnol. 20 (1):17-27. liter, about 0.1 grams per liter to about 4 grams per liter, about Fermentation may be performed at any scale. The expres 0.1 grams per liter to about 5 grams per liter, about 0.1 grams sion systems according to the present invention are useful for per liter to about 6 grams per liter, about 0.1 grams per liter to recombinant protein expression at any scale. Thus, e.g., 55 about 7 grams per liter, about 0.1 grams per liter to about 8 microliter-scale, milliliter scale, centiliter scale, and deciliter grams per liter, about 0.1 grams per liter to about 9 grams per scale fermentation Volumes may be used, and 1 Liter scale liter, about 0.1 grams per liter to about 10 grams per liter, and larger fermentation Volumes can be used. about 1 gram per liter to about 2 grams per liter, about 2 grams In embodiments, the fermentation volume is at or above per liter to about 3 grams per liter, about 3 grams per liter to about 1 Liter. In embodiments, the fermentation volume is 60 about 4 grams per liter, about 4 grams per liter to about 5 about 1 liter to about 100 liters. In embodiments, the fermen grams per liter, about 5 grams per liter to about 6 grams per tation volume is about 1 liter, about 2 liters, about 3 liters, liter, about 6 grams per liter to about 7 grams per liter, about about 4 liters, about 5 liters, about 6 liters, about 7 liters, about 7 grams per liter to about 8 grams per liter, about 8 grams per 8 liters, about 9 liters, or about 10 liters. In embodiments, the liter to about 9 grams per liter, or about 9 grams per liter to fermentation volume is about 1 liter to about 5 liters, about 1 65 about 10 grams per liter. In embodiments, the soluble recom liter to about 10 liters, about 1 liter to about 25 liters, about 1 binant protein yield is about 1 gram per liter to about 3 grams liter to about 50 liters, about 1 liter to about 75 liters, about 10 per liter, about 2 grams per liter to about 4 grams per liter, US 8,455,218 B2 23 24 about 3 grams per liter to about 5 grams per liter, about 4 In embodiments, the recombinant hCG-CSF protein com grams per liter to about 6 grams per liter, about 5 grams per prises an 175 amino acid protein with an N-terminal methion liter to about 7 grams per liter, about 6 grams per liter to about ine (Met-G-CSF). In embodiments, the recombinant hC-CSF 8 grams per liter, about 7 grams per liter to about 9 grams per protein is not glycosylated. liter, or about 8 grams per liter to about 10 grams per liter. In 5 Solubility and Activity embodiments, the soluble recombinant protein yield is about The “solubility” and “activity” of a protein, though related qualities, are generally determined by different means. Solu 0.5 grams per liter to about 4 grams per liter, about 1 gram per bility of a protein, particularly a hydrophobic protein, indi liter to about 5 grams per liter, about 2 grams per liter to about cates that hydrophobic amino acid residues are improperly 6 grams per liter, about 3 grams per liter to about 7 grams per located on the outside of the folded protein. Protein activity, liter, about 4 grams per liter to about 8 grams per liter, about 10 which can be evaluated using different methods, e.g., as 5 grams per liter to about 9 grams per liter, or about 6 grams described below, is another indicator of proper protein con per liter to about 10 grams per liter. In embodiments, the formation. “Soluble, active, or both' as used herein, refers to extracted protein yield is about 0.5 grams per liter to about 5 protein that is determined to be soluble, active, or both soluble grams per liter, about 0.5 gram per liter to about 10 grams per and active, by methods known to those of skill in the art. liter, about 1 grams per liter to about 6 grams per liter, about 15 Activity Assay 2 grams per liter to about 7 grams per liter, about 3 grams per Assays for evaluating hCG-CSF activity are know in the art liter to about 8 grams per liter, about 4 grams per liter to about and can include binding to recombinant human granulocyte 9 grams per liter, or about 5 grams per liter to about 10 grams colony stimulating factor receptor (GCSF-R). A binding per liter. assay is described in Example 1. In embodiments, the amount of recombinant hCG-CSF pro In embodiments, activity is represented by the percent tein comprising an N-terminal methionine (Met-G-CSF) active protein in the extract Supernatant as compared with the detected in the soluble fraction is about 10% to about 100% of total amount assayed. This is based on the amount of protein the amount of the total recombinant hCG-CSF produced. In determined to be active by the assay relative to the total embodiments, this amount is about 10%, about 15%, about amount of protein used in assay. In other embodiments, activ 20%, about 25%, about 30%, about 35%, about 40%, about 25 ity is represented by the % activity level of the protein com pared to a standard, e.g., native protein. This is based on the 45%, about 50%, about 55%, about 60%, about 65%, about amount of active protein in Supernatant extract sample rela 70%, about 75%, about 80%, about 85%, about 90%, about tive to the amount of active protein in a standard sample 95% or about 99%, or about 100% of the amount of the total (where the same amount of protein from each sample is used recombinant hCG-CSF produced. In embodiments, this in assay). amount is about 10% to about 20%, 20% to about 50%, about 30 In embodiments, about 40% to about 100% of the recom 25% to about 50%, about 25% to about 50%, about 25% to binanthC-CSF protein comprising an N-terminal methionine about 95%, about 30% to about 50%, about 30% to about (Met-G-CSF), is determined to be active. In embodiments, 40%, about 30% to about 60%, about 30% to about 70%, about 40%, about 50%, about 60%, about 70%, about 80%, about 35% to about 50%, about 35% to about 70%, about 35% about 90%, or about 100% of the recombinant hCG-CSF pro to about 75%, about 35% to about 95%, about 40% to about 35 tein is determined to be active. In embodiments, about 40% to 50%, about 40% to about 95%, about 50% to about 75%, about 50%, about 50% to about 60%, about 60% to about about 50% to about 95%, about 70% to about 95%, or about 70%, about 70% to about 80%, about 80% to about 90%, 80 to about 100% of the amount of the total recombinant about 90% to about 100%, about 50% to about 100%, about hC-CSF produced. 60% to about 100%, about 70% to about 100%, about 80% to In embodiments, the amount of soluble recombinant hC 40 about 100%, about 40% to about 90%, about 40% to about CSF protein comprising an N-terminal methionine (Met-G- 95%, about 50% to about 90%, about 50% to about 95%, CSF) produced is about 0.1% to about 50% of the total about 50% to about 100%, about 60% to about 90%, about soluble protein produced in a culture. In embodiments, this 60% to about 95%, about 60% to about 100%, about 70% to amount is more than about 0.1%, 0.5%, 1%. 5%, 10%, 15%, about 90%, about 70% to about 95%, about 70% to about 20%, 25%, 30%, 40%, 45%, or 50% of the total soluble 45 100%, or about 70% to about 100% of the recombinant hCG protein produced in a culture. In embodiments, this amount is CSF protein comprising an N-terminal methionine (Met-G- CSF) is determined to be active. about 0.1%, 0.5%, 1%. 5%, 10%, 15%, 20%, 25%, 30%, In other embodiments, about 75% to about 100% of the 40%, 45%, or 50% of the total soluble protein produced in a recombinant hCG-CSF protein comprising an N-terminal culture. In embodiments, this amount is about 5% to about methionine (Met-G-CSF) is determined to be active. In 50%, about 10% to about 40%, about 20% to about 30%, 50 embodiments, about 75% to about 80%, about 75% to about about 1% to about 20%, about 5% to about 25%, about 1% to 85%, about 75% to about 90%, about 75% to about 95%, about 10%, about 10% to about 20%, about 20% to about about 80% to about 85%, about 80% to about 90%, about 80% 30%, about 30% to about 40%, or about 40% to about 50% of to about 95%, about 80% to about 100%, about 85% to about the total soluble protein produced in a culture. 90%, about 85% to about 95%, about 85% to about 100%, In embodiments, the amount of soluble recombinant hC 55 about 90% to about 95%, about 90% to about 100%, or about CSF protein comprising an N-terminal methionine (Met-G- 95% to about 100% of the recombinanthC-CSF protein com CSF) produced is about 0.1% to about 50% of the dry cell prising an N-terminal methionine (Met-G-CSF) is deter weight (DCW). In embodiments, this amount is more than mined to be active. about 0.1%, 0.5%, 1%. 5%, 10%, 15%, 20%, 25%, 30%, EXAMPLE 40%, 45%, or 50% of DCW. In embodiments, this amount is 60 about 0.1%, 0.5%, 1%. 5%, 10%, 15%, 20%, 25%, 30%, Example 1 40%, 45%, or 50% of DCW. In embodiments, this amount is about 5% to about 50%, about 10% to about 40%, about 20% Screening for Met-G-CSF Expression to about 30%, about 1% to about 20%, about 5% to about 25%, about 1% to about 10%, about 10% to about 20%, about 65 Design of a Synthetic G-CSF Gene for Optimal Expression. 20% to about 30%, about 30% to about 40%, or about 40% to The following criteria were used for the design of the about 50% of the total soluble protein produced in a culture. synthetic G-CSF gene that was optimized for high-level pro US 8,455,218 B2 25 26 duction in Pseudomonas fluorescens. A DNA sequence cod strands of DNA from promoter through transcriptional termi ing for G-CSF (FIG. 6) was designed to reflect appropriate nator. Reactions consisted of2 uL of sequencing premix, 1 ul codon usage for Pfluorescens strain MB101. A DNA region of 6.4 uM primer, 50 fmol of DNA template, 3 ul. 5x buffer, containing a unique restriction enzyme site (SapI) was added and H2O to adjust volume to 20 uL. Sequencing reactions upstream of the GCSF coding sequence designed for direct were purified using Sephadex G-50 (Sigma, 55897) and fusion inframe with the secretion leader present in the expres loaded into the ABI3100 sequencer. Sequence data were sion vector. A DNA region containing 3 stop codons and a assembled and analyzed using Sequencher software (Gene unique restriction enzyme site (Sap) was added downstream Codes). of the coding sequence. All gene-internal ribosome binding Growth and Expression in 96-Well Format sites which matched the pattern aggaggtins-odtg with 2 or 10 The effect of a variety of expression plasmids and host fewer mismatches were removed to avoid potentially trun strains on Met-G-CSF expression was evaluated at the cated protein products. Stretches of 5 or more C or 5 or more 96-well scale. Nineteen secretion leaders fused to Met-G- G nucleotides were eliminated to avoid RNA polymerase CSF constructs were tested in Pfluorescens DC454. slippage. Strong gene-internal stem-loop structures, espe 15 Cells were electroporated with indicated plasmids, resus cially those covering the ribosome binding site, were pended in HTP growth medium with trace minerals and 5% removed. The synthetic gene was produced by DNA2.0 Inc. glycerol and then transferred to 96-well deep well plates with (Menlo Park, Calif.). 400 uM9 salts 1% glucose medium and trace elements. The Synthesis the Met-G-CSF Gene by PCR Amplifications 96-well plates were incubated at 30°C. with shaking for 48 hours. Ten microliters of these seed cultures were transferred Standard cloning methods were used in the construction of into triplicate 96-well deep well plates, each well containing cytoplasmic and periplasmic Met-GCSF expression plas 500 ul of HTP medium, supplemented with trace elements mids. PCR products were generated using PfusionTM high and 5% glycerol, and incubated as before for 24 hours. Iso fidelity PCR kit (New England Biolabs, F-553L) and p201: propyl BD 1 thiogalactopyranoside (IPTG) was added to each 25893 as template with combinations of the primers GCSF 25 well for a final concentration of 0.3 mM to induce the expres cyto-forward, GCSF cyto-reverse for cytoplasmic Met sion of GCSF and temperature was reduced to 25°C. Twenty GCSF construct; GCSF-peri-forward and GCSF cyto-reverse four hours after induction, cells were normalized to for the periplasmic constructs (Table 4). The PCR products OD600–15 using PBS in a volume of 400 ul using the Biomek were SapI digested and cloned into the rapid cloning vectors. liquid handling station (Beckman Coulter). Samples were Insert and vectors were ligated overnight with T4DNA ligase 30 frozen for later processing by Sonication and centrifugation to (New England Biolabs, M0202S), and electroporated into generate soluble and insoluble fractions. competent Pfluorescens DC454 cells and transformants Sample Preparation were selected on M9 glucose agar plates. Positive clones were Soluble fractions were prepared by sonication followed by selected and sequence-confirmed on both Strands using prim centrifugation. Normalized culture broth samples (400 uL) ers in Table 2. The resulting cytoplasmic expression plasmid 35 were sonicated with the Cell Lysis Automated Sonication was named p529-013, which was transformed into 16 System (CLASS, Scinomix) with a 24 probe tip horn. The selected Pfluorescens host strains in 96-well format. lysates were centrifuged at 14,000xrpm for 20 minutes (4 C.) and the supernatants collected (soluble fraction). Further TABLE 4 dilutions of soluble samples for biolayer interferometry 40 analysis were performed in ForteBios Sample Diluent (PN, Primers used in current study described below). GCSF Binding Biolayer Interferometry SEQ ID The lyophilized recombinant human granulocyte colony Name Length Oligo Sequence NO : stimulating factor receptor (GCSF-R) (R&D Systems, cathi 45 381GR/CF) was reconstituted in PBS buffer (Sigma, P3813) GCSF-peri 34 ATATGCTCTTCAGCCATGA 1. to 0.5 mg/mL and biotinylated using NHS-LC-LC-biotin forward CTCCTCTGGGTCCTG (Pierce, 21343) according to the method described in Forte GCSF Cyto- 37 ATATGCTCTTCTGAAGTGA 2 Bio Technical Note: “Biotinylation of Protein for Immobili rewerse CTCTCGAGCTATTATCAC zation onto Streptavidin Sensors.” The biosensors (Streptavi 50 GCSF Cyto- 31 ATATGCTCTTCAATGACTC 3 din High Binding FA Biosensors, ForteBio, 18-0009) were forward CTCTGGGTCCTG hydrated in 1x kinetics buffer (10-fold dilution of 10x Kinet ics Buffer, ForteBio, 18-5032 into PBS) for at least 10 min GCSF-Mid- 19 GCAGGCCTTGGAAGGCATC 4. utes. The sensors were loaded with 10 ug/mL biotinylated forward GCSF-R (b-GCSF-R) diluted into sample diluent (ForteBio, GCSF-Mid 19 GATGCCTTCCAAGGCCTGC 5 55 18-1000) for at least 2 hours at room temperature or overnight rewerse at 4° C. The soluble fraction of the strain array samples were Term 18 GCTGCCGCACAGCTCCAT 6 diluted 20-fold into 1x kinetics buffer. Standards were diluted Ptac 22 CCGATGATCGGTAAATACC 7 into Pfluorescens DC432 null soluble fraction. Samples and GAT 60 standards were loaded at a volume of 100 uL into half area plates (E&K Scientific, EK-78076). The b-GCSF-R loaded sensors were soaked in 1x kinetics buffer for ~5 minutes to DNA Sequencing rinse away unbound ligand, and then pre-equilibrated for Clones were analyzed by sequencing using BigDye R. Ter 40-60 minutes in a dilution of null soluble fraction. The minator v3.1 Cycle Sequencing Kit (Applied BioSystems, 65 sample plate was pre-warmed at 30° C. in the Octet instru 4337455). Vector primers flanking the DNA insert and 2 ment for 10 minutes prior to initiating the assay. The samples insert specific primers (Table 4) were used to confirm the both were read at 200 rpm, 30° C., for 120 sec, and quantitation US 8,455,218 B2 27 28 was calculated from a standard curve at 12, 6, 3, 1.5, 0.75, protein to the periplasm where it may be recovered in the 0.375, 0.188, and in some cases as low as 0.094 g/ml. properly folded and active form. The gene was cloned into the Intact Mass Analysis of Target Protein expression vectors as described, and the sequences were con For HTP samples, 50 ul soluble lysates were mixed with 50 firmed from the promoter through the transcriptional termi ul of 5.4 MGu-HCl, 100 mMDTT and heated at 37° C. for 10 nator. A representative plasmid map (p529-016) is shown in min. The mixture was centrifuged at 18,000 g for 5 min. at FIG. 3. Expression of the G-CSF was driven from the Ptac room temperature (R.T.), and transferred to an autosample promoter, and translation initiated from a high activity ribo vial with a 200 ul polypropylene insert (Agilent, 5.182-0549). some binding site (RBS). The resulting 19 plasmids were For mini-bioreactor (MBR) samples, -75 ul of MBR transformed into P. fluorescens host strains as described lysates were subjected to acetone precipitation. Briefly, 6 10 below. Folding modulators, when present, were encoded on a volumes of cold acetone were added to the MBR lysates, second plasmid and expression driven by a mannitol induc Vortexed and incubated on ice for 20 min. The mixtures were ible promoter. then centrifuging at 18,000 g for 5 min. at R.T. The pellets were washed with cold 85% acetone and centrifuged as TABLE 5 above, and the pellets were air-dried for 10 min. The pellets 15 Secretion leaders tested for the Met-G-CSF were then solubilized in 50 ul of 5.4 MGu-HCl, 100mMDTT Construct and heated at 37°C. for 10 min. Fifty ul of HO was added to SEQ each sample, the mixture was centrifuged as above, and ID Supernatant was transferred to autosample vials. Leader Sequence NO : The above HTP or MBR lysates were subjected to LC-MS analysis using an interconnected autosampler, column heater, Pbp KLKRLMAAMTFWAAGWATWNAWA 8 UV detector, and HPLC (Agilent 1100) coupled to a Q-T of micro mass spectrometer (Waters) with an electrospray inter DsbA. RNLILSAALWTASLFGMTAOA 9 face. A C8 column (Zorbax 5um, 300SE-C8, 2.1x150 mm, Azul FAKLVAVSLLTLASGOLLA O Agilent) fitted with a guard column (Zorbax 5um,300SEB-C8, 25 2.1 x 12.5 mm, Agilent) was used for separation. The HPLC LAO QNYKKFLLAAAVSMAFSATAMA 1. buffers used were buffer A (0.1% formic acid) and buffer B Ibp - S31A IRDNRLKTSLLRGLTITLLSLTLLSPAAHA 2 (90% acetonitrile 0.1% formic acid). After the column was equilibrated at 5% buffer B and the sample was loaded, the ToB RNLLRGMLWWICCMAGIAAA. 3 on-column sample was Subjected to one of two different 30 reversed phase gradients. For the “70 min. C8 method, the Tpr NRSSALLLAFWFLSGCQAMA 4. column was washed at 5% B for 10 min., and then a 40 min. Ttg2C MONRTVEIGWGLFLLAGILALLLLALRVSGLSA 15 gradient from 5% to 60% B was used. For the “35 min. C8 method.” after loading at 5% B, a steep linear gradient wa Flg.I KFKQLMAMALLLALSAVAQA 6 developed to 53% B over 10 min. Subsequently, a shallow 35 Cup C2 PPRSIAACLGLLGLLMATOAAA 7 gradient from 53% to 63% over 10 min. was developed. For both methods, after the gradient, the solvent was brought to CupB2 FRTLLASLTFAWIAGLPSTAHA 8 100% B for 5 min., ending with 5% B for 5 min. The retention Cup.A2 SCTRAFKPLLLIGLATLMCSHAFA 9 time of G-CSF was -51.5 min. and -19.5 min. for the “70 min. C8 method” and the "35 min. C8 method.” respectively. 40 NikA RIAALPLLLAPLFIAPMAWA 2O Another column was used and method developed in hopes of reducing the clogging/fouling observed from the analysis CopA. SHFDLGRRRVMOAVGAGLLLPGLAPAVIA 21 of MBR lysates. A CN column (Zorbax 5um, 300SE-CN, Pbp-A2 OV KLKRLMAAMTFWAAGWATWNAWA 22 2.1 x 150 mm, Agilent) fitted with a C3 guard column (Zorbax 5um, 300SEB-C3, 2.1x12.5 mm, Agilent) was used for sepa 45 DSC RLTOIIAAAAIALWSTFALA 23 ration. ALC method was developed for loading of lysates at 20% B, followed by a gradient of 20% to 40% Bover 10 min. Bce STRIPRRQWLKGASGLLAAASLGRLANREARA 24 and followed by another gradient of 40% to 60% B over 20 MooD HRRNLLKASMAIAAYTGLSASGLLAAQAWA 25 min. The column was then brought to 100% B for 5 min., and OprF KLKNTLGLAIGSLIAATSFGWLA 26 ended with 20% B for 5 min. The retention time of G-CSF for 50 this “40 min. CN method is -19.5 min. UV absorbance was collected from 180-500 nm, prior to Small Scale Expression of G-CSF in Pfluorescens MS. The MS source was used in positive mode at 2.5 kV. MS The effect of a variety of expression plasmids and host scans were carried out using a range of 400-2400 m/z at 2 strains on Met G-CSF expression was evaluated. No soluble scans per second. MS and UV data were analyzed using 55 Met G-CSF was detected from strains carrying the cytoplas MassLynx software (Waters). UV chromatograms of MS mic construct p529-013, as determined by BLI. Soluble peri total ion current (TIC) chromatograms were generated. The plasmic expression, up to 250 mg/L, was detected in Strains MS spectra of the target peaks were Summed. These spectra carrying a variety of Secretion leader constructs (FIG. 1). were deconvoluted using MaxEnt 1 (Waters) scanning for a Soluble G-CSF expression up to 250 mg/L was detected in molecular weight range of 14,000-24,000 at a resolution of 60 strains carrying a variety of secretion leader constructs (FIG. 0.5 Da per channel. 1). For selected higher expression strains, Western blot and/or Construction of G-CSF Expression Strains intact mass analysis were performed for target identification An optimized human gcSf gene was designed and synthe and evaluation of leader processing. The results indicated sized for expression in Pfluorescens as described above. seven secretion leaders (Pbp, Flg.I, DsbA, LAO, Cup A2. Ibp Plasmids were constructed carrying the optimized gcSf gene 65 and Pbp-A20V) were efficiently processed (data not shown). fused to 19 different Pfluorescens secretion leaders (Retal Western blot and intact mass (data not shown) analysis lack et al., 2007) (Table 5). The secretion leaders target the were performed on selected strains. The results indicated six US 8,455,218 B2 29 30 secretion leaders: Pbp, FlgI, DsbA, LAO, Cup A2, and Ibp. TABLE 8-continued were efficiently processed. Another leader, Pbp-A20V, was efficiently processed as determined by intact mass analysis Top G-CSF Expression Strains Analyzed by LC-MS. (data not shown). Strain Host Plasmi Yield (mg/L) des-Met(%) TABLE 6 CS529-765 MID472O 529-017 281 S.OO CSS29-563 MID4692 b529-017 3O2 S.OO CS529-770 MID4739 b529-017 309 S.OO Met-G-CSF Expression Strains. CSS29-828 DC1067 S29-O2O 336 S.OO Signal Plasmid CSS29-716 DC1030 S29-017 353 S.OO Sequence No. 10 CSS29-564 MID4693 b529-017 3S4 S.OO CS529-753 DC487 529-017 425 S.OO Pbp p529-015 CS529-515 DCSO8 529-017 430 S.OO DsbA. p529-016 CS529-747 DC1063 b529-017 437 S.OO Lao p529-017 CSS29-829 DC1076 529-O2O 457 S.OO Ibp-S31A p529-018 CS529-715 DC1029 .529-017 474 S.OO Flg.I p529-019 15 CSS29-81.8 DC1074 b529-O2O 545 S.OO Cup A2 p529-020 CS529-776 DC1098 529-017 227 S.40 Pbp-A2OV p529-021 CS529-571 MID4707 S29-017 299 6.OO CSS29-78O DCO859 p529-O2O 51 O.OO CSS29-806 DC1068 b529-O2O 82 O.OO CSS29-788 DC102O 529-O2O 129 O.OO Screening of the Protease Deletions CSS29-789 DC1021 529-O20 146 O.OO All available protease deletion hosts of Pfluorescens were CS529-745 DC954 529-017 250 O.OO screened for minimal des-Met G-CSF production. Plasmids CS529-785 DCO977 529-O2O 258 O.OO CS529-556 MID4764 b529-017 274 O.OO carrying Met-G-CSF gene fused to the secretion leaders CSS29-711 DC1 O2S 529-017 286 O.OO (Table 6) were transformed into the protease deletion library. CSS29-782 DCO95S 529-O2O 3O2 O.OO The resulting strains were grown for 24 hours, induced with CS529-751 DC485 529-017 304 O.OO IPTG and harvested 24 hours post induction. 25 CSS29-561 MID4690 b529-017 316 O.OO CSS29-762 MID4717 S29-017 366 O.OO CS529-574 MID4710 b529-017 383 O.OO TABLE 7 CS529-756 DC490 529-017 402 O.OO CSS29-796 DC1030 S29-O2O 409 O.OO Selected plasmids for Screening the protease deletion host library. CSS29-744 DC441 529-017 414 O.OO 30 CSS29-526 DC987 529-017 418 O.OO Plasmid Signal Sequence CS529-565 MID4694 b529-017 433 O.OO CSS29-82O DC674 529-O20 S8O O.OO p529-017 Lao CS529-703 DCO956 S29-017 208 S.OO p529-018 Ibp-S31A CSS29-783 DCO956 S29-O2O 255 S.OO p529-020 cup A2 CSS29-853 DC1095 S29-O2O 312 S.OO 35 CSS29-560 MID4689 b529-017 355 S.OO CSS29-519 DC52O 529-017 377 S.OO Analysis of Protein Quantity and Quality CSS29-544 MID4749 b529-017 383 S.OO After harvest, samples were normalized to OD600 of 15. CSS29-543 MID2O78 b529-017 397 S.OO Cells were sonicated and separated into soluble and insoluble CSS29-541 MID2O74 b529-017 449 S.OO CSS29-8SO MID4739 b529-O20 255 2O.OO fractions. The soluble protein expression was analyzed by CSS29-554 MID4761 b529-017 319 2O.OO BLI binding to the GCSF-R. The soluble G-CSF yield ranged 40 CSS29-534 DC1041 529-017 331 2O.OO from non-detectable to 470 mg/L. The high G-CSF express CS529-517 DCS11 529-017 353 2O.OO ing strains were selected for quality evaluation by intact mass CSS29-545 MID47SO 529-017 358 2O.OO CS529-535 DC1042 529-017 359 2O.OO analysis. Table 8 shows the selected G-CSF expression strains CSS29-514 DC507 529-017 374 2O.OO and their corresponding LC-MS analysis. The des-Met (%) is CS529-525 DC983 529-017 379 2O.OO the percentage of des-Met relative to the intact Met-G-CSF 45 CSS29-712 DC1026 S29-017 350 2S.OO amount. The host strain MID4697, which contains an inser CSS29-786 DC1010 529-O2O 196 3O.OO CSS29-840 DC977 529-O20 242 3O.OO tionally inactivated prtB gene, was identified as having the CSS29-546 MID4751 b529-017 336 3O.OO lowest level of des-Met GCSF. CS529-558 MID4687 S29-017 348 3O.OO CSS29-834 DC488 529-O20 372 3S.OO TABLE 8 50 CSS29-718 DC1033 S29-017 164 40.OO CSS29-659 DC696 529-O20 192 4S.OO Top G-CSF Expression Strains Analyzed by LC-MS. CSS29-598 DCS18 529-O20 175 SO.OO CS529-717 DC1032 529-017 151 8O.OO Strain Host Plasmi Yield (mg/L) des-Met(%) CSS29-719 DC1034 b529-017 132 1OOOO

CSS29-568 MID4697 S29-017 247 O.10 55 CSS29-648 MID4697 S29-O20 297 O.30 Construction of a Clean Knock Out of prtB Gene CSS29-700 DCO859 p529-017 173 O.SO CS529-750 DC469 529-017 380 O.SO The host MID4697 showed only up to 0.3% des-Met CSS29-708 DC102O 529-017 281 1.00 G-CSF product from the cell lysates at the 96-well plate scale CSS29-726 DC1068 b529-017 204 140 (Table 8). Strain MID4697 contains an insertional mutation CSS29-709 DC1021 529-017 146 160 of rxfo8627, which encodes the extracellular serine protease CSS29-830 DC469 529-O20 321 1.70 60 CSS29-518 DCS18 529-017 335 2.70 PrtB, in a AaprA Pfluorescens strain. Strain MID4697 con CSS29-790 DC1023 S29-O2O 39 3.00 tains an antibiotic (kanamycin) resistant marker in its CS529-705 DCO977 529-017 191 3.20 genome, which is not desirable. Therefore, anrxfo8627 dele CS529-775 DC1097 529-017 189 S.OO tion strain was constructed. CS529-707 DC1011 529-017 193 S.OO CS529-735 DC106S 529-017 229 S.OO 65 A complete rxf)8627 gene deletion strain was constructed CS529-731 DC1084 b529-017 233 S.OO to inactivate the annotated extracellular protease Rxfo8627 from the genome. A deletion plasmid pl)OW6800 was con US 8,455,218 B2 31 32 structed by PCR amplification of two DNA fragments flank the culture from each fermentor was harvested by centrifu ing the rxf)8627 region. The two fragments were subse gation and the cell pellet frozen at -80°C. quently fused using the splicing by overlap extension PCR The exemplifying fermentation cultures, induced at 80 OD method. The fused DNA fragments were then ligated into the with 0.24 mMIPTG, pH and temperature setpoints adjusted SrfI site of vectorp)OW1261 to create the deletion plasmid 5 to 6.0 of 28.5°C., respectively, resulted in 0.35 g/L of soluble, pDOW6800. The insert of the deletion plasmid was con active Met G-CSF (FIGS. 4 and 5). firmed by DNA sequencing. The deletion of rxf)8627 gene Evaluation of G-CSF Production in prtB Deletion Host was created by cross-in cross-out allele exchange as Strains described (Schneider, et al., 2005). A clean deletion of The quality of the product from the newly constructed rxfo8627 was constructed in the wild type and AaprA P 10 strains (clean deletion of prtB) (Table 9) was evaluated by fluorescens strain backgrounds, resulting in strains MID5093 LC-MS analysis following expression at the 4 mL fermenta and MID5103 respectively. tion scale; Met-GCSF was detected, while no des-Met G-CSF product was observed. FIG. 2 shows representative strains TABLE 9 analyzed by LC-MS. 15 G-CSF In Vitro Cell Proliferative Activity Assay New Strains Constructed for Expression of Met-G-CSF A G-CSF activity assay was performed using the myeloid leukemia M-NFS-60 proliferation method following the pro Strain Name Signal Sequence Plasmid Host tocol described by Hammerling et al. (1995). The M-NFS-60 CSS29-900 pbp 529-O15 MIDSO93 cell line was purchased from the American Type Culture CSS29-901 disbA b529-O16 MIDSO93 Collection (ATCC, CRL-1838). The stimulatory effect of CSS29-902 Lao 529-017 MIDSO93 CSS29-903 Ibp-S31A 529-018 MIDSO93 G-CSF was measured and compared with that of NeupogenR) CSS29-904 Flg.I 529-019 MIDSO93 using a colorimetric assay utilizing a rapid cell proliferation CSS29-90S cup A2 529-O20 MIDSO93 assay kit (Calbiochem, QIA127). The purified Met-G-CSF CSS29-906 Pbp-A2OV 529-021 MIDSO93 induced NFS-60 cell proliferation, with a 50% Effective Dose CSS29-907 pbp 529-O15 MIDS103 25 CSS29-908 disbA b529-O16 MIDS103 (ED50) of 35 pg/mL. The dose responsive curve of purified CSS29-909 Lao 529-017 MIDS103 G-CSF was very similar to that of Neupogen R in the M-NFS CSS29-910 Ibp-S31A 529-018 MIDS103 60 proliferation assay (FIG. 8). As illustrated in FIG. 8, pro CSS29-911 Flg.I 529-019 MIDS103 liferation of the murine myeloblastic cell line NFS-60 was CSS29-912 cup A2 529-O20 MIDS103 CSS29-913 Pbp-A2OV 529-021 MIDS103 measured. The concentration of CS5329-901-produced 30 G-CSF (open circles) and Neupogen R. (closed squares) is shown on the X-axis, and the absorbance at 450 nm repre Strain Evaluation in Minibioreactors (MBR) senting cell proliferation is shown on the Y-axis. The error The expression strains were evaluated for production of bars represent the standard error of three replicates for each soluble G-CSF in 9 different fermentation conditions using point. the L-24TM micro-bioreactor (MBR: Applikon Biotechnol 35 ogy), a 24-well mini-bioreactor system designed to indepen REFERENCES dently monitor and control pH, temperature and DO in each well. Fractional factorial DOE experiments were conducted Covalt, J. C., Jr., Cao, T. B., Magdaroag, J. R. Gross, L.A. & to examine effects of varying multiple fermentation param Jennings, P. A. (2005). Temperature, media, and point of eters. The MBR single-use polystyrene cassette had 24 wells 40 induction affect the N-terminal processing of interleukin and operated at a 4-mL working Volume. For experiments 1beta. Protein Expr Purif 41, 45-52. conducted with oxygen as the sparging gas, the set points U. Hammerling, R. Kroon, and L. Sjodin, In vitro bioassay were programmed to maintain constant agitation, tempera with enhanced sensitivity for human granulocyte colony ture and pH during the growth phase, and DO control was set stimulating factor. J Pharm Biomed Anal. 13 (1995) 9-20. at 30%. Glycerol was provided at 30-60 g/L in a minimal salts 45 Herman, A. C., Boone, T. C. & Lu, H. S. (1996). Character medium (Riesenberg et al., 1991) in order to support growth ization, formulation, and stability of Neupogen of the cultures to different induction ODs without the need for (Filgrastim), a recombinant human granulocyte-colony Subsequent feed addition. stimulating factor. Pharm Biotechnol 9, 303-28. Production of recombinant Met G-CSF protein in Okabe, M., Asano, M., Kuga, T., Komatsu, Y. Yamasaki, M., Pseudomonas fluorescens Pfenex Expression TechnologyTM 50 Yokoo,Y., Itoh, S., Morimoto, M. & Oka, T. (1990). In vitro strain CS529-901 was successfully achieved in 2 liter fermen and in vivo hematopoietic effect of mutant human granu tors. Multiple fermentation conditions were evaluated result locyte colony-stimulating factor. Blood 75, 1788-93. ing in expression of Met G-CSFup to 0.35 g/L as measured by Retallack, D. M., Schneider, J. C., Mitchell, J., Chew, L. & BLI binding assay. Liu, H. (2007). Transport of heterologous proteins to the Fermentation cultures were grown in 2 liter fermentors 55 periplasmic space of Pseudomonas fluorescens using a containing a mineral salts medium (Riesenberg, et al., 1991). variety of native signal sequences. Biotechnol Lett 29, Culture conditions were maintained at 32° C. and pH 6.5 1483-91 through the addition of aqueous ammonia. Dissolved oxygen Tanaka, H., Tanaka, Y., Shinagawa, K. Yamagishi.Y., Ohtaki, was maintained in excess through increases in agitation and K. & Asano, K. (1997). Three types of recombinant human flow of sparged air and oxygen into the fermentor. Glycerol 60 granulocyte colony-stimulating factor have equivalent bio was delivered to the culture throughout the fermentation to logical activities in monkeys. Cytokine 9, 360-9. maintain excess levels. These conditions were maintained Weston, B., Todd, R. F., 3rd, Axtell, R., Balazovich, K., Stew until the target culture optical density (Asas) for induction art, J., Locey, B.J., Mayo-Bond, L., Loos, P. Hutchinson, was reached, at which time IPTG was added to initiate Met R. & Boxer, L. A. (1991). Severe congenital neutropenia: G-CSF production. The optical density at induction, the con 65 clinical effects and neutrophil function during treatment centration of IPTG, pH and temperature were all varied to with granulocyte colony-stimulating factor. J Lab Clin determine optimal conditions for expression. After 16 hours, Med 117, 282-90. US 8,455,218 B2 33 34 Riesenberg, D.; Schulz, V. Knorre, W. A.; Pohl, H. D.; Korz, substitutions will now occur to those skilled in the art without D.; Sanders, E. A.; Ross, A.; Deckwer, W. D. High cell departing from the invention. It should be understood that density cultivation of Escherichia coli at controlled spe various alternatives to the embodiments of the invention cific growth rate. J. Biotechnol. 1991, 20 (1), 17-27. described herein may be employed in practicing the inven While preferred embodiments of the present invention tion. It is intended that the following claims define the scope have been shown and described herein, it will be obvious to of the invention and that methods and structures within the those skilled in the art that such embodiments are provided by Scope of these claims and their equivalents be covered way of example only. Numerous variations, changes, and thereby.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 34

<21 Os SEQ ID NO 1 &211s LENGTH: 34 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OOs SEQUENCE: 1

atatgct citt cago catgac to citctgggit cotg 34

<21 Os SEQ ID NO 2 &211s LENGTH: 37 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OOs, SEQUENCE: 2

atatgct citt ctdaagtgac totcgagcta titat cac 37

<21 Os SEQ ID NO 3 &211s LENGTH: 31 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OOs SEQUENCE: 3

atatgct citt caatgactic c totgggit cot g 31

<21 Os SEQ ID NO 4 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OOs SEQUENCE: 4

gcaggccttg galaggcatc 19

<21 Os SEQ ID NO 5 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic primer

<4 OOs SEQUENCE: 5

gatgcct tcc aaggcctgc 19 US 8,455,218 B2 35 36 - Continued

<210s, SEQ ID NO 6 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic prlmer

<4 OOs, SEQUENCE: 6 gctg.ccgcac agct coat 18

<210s, SEQ ID NO 7 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic prlmer

<4 OO > SEQUENCE: 7 ccgatgat cq gtaaat accg at 22

<210s, SEQ ID NO 8 &211s LENGTH: 24 212. TYPE: PRT <213s ORGANISM: Pseudomonas fluorescens

<4 OOs, SEQUENCE: 8 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 9 &211s LENGTH: 22 212. TYPE: PRT <213s ORGANISM: Pseudomonas fluorescens

<4 OOs, SEQUENCE: 9 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 10 &211s LENGTH: 2O 212. TYPE: PRT <213s ORGANISM: Pseudomonas fluorescens

<4 OOs, SEQUENCE: 10 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

<210s, SEQ ID NO 11 &211s LENGTH: 23 212. TYPE: PRT <213s ORGANISM: Pseudomonas fluorescens

<4 OOs, SEQUENCE: 11 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 US 8,455,218 B2 37 38 - Continued

<210s, SEQ ID NO 12 &211s LENGTH: 31 212. TYPE: PRT <213s ORGANISM: Pseudomonas fluorescens

<4 OOs, SEQUENCE: 12 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 13 &211s LENGTH: 21 212. TYPE: PRT <213s ORGANISM: Pseudomonas fluorescens

<4 OOs, SEQUENCE: 13 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 14 &211s LENGTH: 21 212. TYPE: PRT <213s ORGANISM: Pseudomonas fluorescens

<4 OOs, SEQUENCE: 14 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 15 &211s LENGTH: 33 212. TYPE: PRT <213s ORGANISM: Pseudomonas fluorescens

<4 OOs, SEQUENCE: 15 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 16 &211s LENGTH: 21 212. TYPE: PRT <213s ORGANISM: Pseudomonas fluorescens

<4 OOs, SEQUENCE: 16 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 17 &211s LENGTH: 23 212. TYPE: PRT <213s ORGANISM: Pseudomonas fluorescens US 8,455,218 B2 39 40 - Continued <4 OOs, SEQUENCE: 17 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 18 &211s LENGTH: 24 212. TYPE: PRT <213s ORGANISM: Pseudomonas fluorescens

<4 OOs, SEQUENCE: 18 Met Lieu. Phe Arg Thr Lieu. Lieu. Ala Ser Lieu. Thir Phe Ala Wall Ile Ala 1. 5 1O 15

Gly Leu Pro Ser Thr Ala His Ala 2O

<210s, SEQ ID NO 19 &211s LENGTH: 25 212. TYPE: PRT <213s ORGANISM: Pseudomonas fluorescens

<4 OOs, SEQUENCE: 19 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 2 O &211s LENGTH: 21 212. TYPE: PRT <213s ORGANISM: Pseudomonas fluorescens

<4 OOs, SEQUENCE: 2O Met Arg Lieu Ala Ala Lieu Pro Lieu. Lieu. Lieu Ala Pro Lieu. Phe Ile Ala 1. 5 1O 15

Pro Met Ala Wall Ala 2O

<210s, SEQ ID NO 21 &211s LENGTH: 30 212. TYPE: PRT <213s ORGANISM: Pseudomonas fluorescens

<4 OOs, SEQUENCE: 21 Met Ser His Phe Asp Leu Gly Arg Arg Arg Val Met Glin Ala Val Gly 1. 5 1O 15

Ala Gly Lieu. Lieu Lleu Pro Gly Lieu Ala Pro Ala Wall Ile Ala 2O 25 3O

<210s, SEQ ID NO 22 &211s LENGTH: 24 212. TYPE: PRT <213s ORGANISM: Pseudomonas fluorescens

<4 OOs, SEQUENCE: 22 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 23 US 8,455,218 B2 41 42 - Continued

&211s LENGTH: 21 212. TYPE: PRT <213s ORGANISM: Pseudomonas fluorescens

<4 OOs, SEQUENCE: 23 Met Arg Lieu. Thr Glin Ile Ile Ala Ala Ala Ala Ile Ala Lieu Wal Ser 1. 5 1O 15

Thir Phe Ala Lieu. Ala 2O

<210s, SEQ ID NO 24 &211s LENGTH: 33 212. TYPE: PRT <213s ORGANISM: Pseudomonas fluorescens

<4 OOs, SEQUENCE: 24 Met Ser Thr Arg Ile Pro Arg Arg Glin Trp Lieu Lys Gly Ala Ser Gly 1. 5 1O 15

Lieu. Lieu Ala Ala Ala Ser Lieu Gly Arg Lieu Ala Asn Arg Glu Ala Arg 2O 25 3O

Ala

<210s, SEQ ID NO 25 &211s LENGTH: 31 212. TYPE: PRT <213s ORGANISM: Pseudomonas fluorescens

<4 OOs, SEQUENCE: 25 Met His Arg Arg ASn Lieu. Lieu. Lys Ala Ser Met Ala Ile Ala Ala Tyr 1. 5 1O 15 Thr Gly Lieu Ser Ala Ser Gly Lieu. Lieu Ala Ala Glin Ala Trp Ala 2O 25 3O

<210s, SEQ ID NO 26 &211s LENGTH: 24 212. TYPE: PRT <213s ORGANISM: Pseudomonas fluorescens

<4 OOs, SEQUENCE: 26 Met Lys Lieu Lys Asn. Thir Lieu. Gly Lieu Ala Ile Gly Ser Lieu. Ile Ala 1. 5 1O 15

Ala Thr Ser Phe Gly Val Lieu. Ala 2O

<210s, SEQ ID NO 27 &211s LENGTH: 525 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polynucleotide 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) . . (525)

<4 OOs, SEQUENCE: 27 atg act cot ctd ggit cot gca agt agt ctd cog caa agt titt ct c ct g 48 Met Thr Pro Leu Gly Pro Ala Ser Ser Lieu Pro Glin Ser Phe Lieu. Lieu. 1. 5 1O 15 aag to Ctg gala Cag gtg cgc aaa att cag ggc gac ggc gca gca Ctg 96 Lys Cys Lieu. Glu Glin Val Arg Lys Ile Glin Gly Asp Gly Ala Ala Lieu. 2O 25 3O

Cag gala aaa citg to gig acc tat aag ttgtc. cac ccc gala gala Ctg 144 Glin Glu Lys Lieu. Cys Ala Thr Tyr Lys Lieu. Cys His Pro Glu Glu Lieu. US 8,455,218 B2 43 44 - Continued

35 4 O 45 gtg ctg. Ctg ggc cat agc ctgggg att CC a tigg gcg ccg ctg. tcg tcc 192 Val Lieu. Lieu. Gly His Ser Lieu. Gly Ile Pro Trp Ala Pro Lieu. Ser Ser SO 55 6 O tgt colt agt caa goc ttg caa ttg gcc ggit togc ctic ticg caa citg cat 24 O Cys Pro Ser Glin Ala Lieu. Glin Lieu Ala Gly Cys Lieu. Ser Glin Lieu. His 65 70 7s 8O agc ggc ctg. itt C Ctg tac Caa ggc ctg. Ctg cag gcc ttg gala ggc at C 288 Ser Gly Lieu. Phe Lieu. Tyr Glin Gly Lieu. Lieu. Glin Ala Lieu. Glu Gly Ile 85 90 95 tcc ccg gaa citg ggc ccg acg Ctg gat acc ctg caa citg gaC gta gca 336 Ser Pro Glu Lieu. Gly Pro Thr Lieu. Asp Thir Lieu. Glin Lieu. Asp Val Ala 1OO 105 11 O gat tt C gcc acg acc atc tig cag cag atg gaa gala Ctg ggc atg gCC 384 Asp Phe Ala Thr Thr Ile Trp Glin Gln Met Glu Glu Lieu. Gly Met Ala 115 12 O 125 ccg gcc ct C cag C cc acg caa ggc gcg atg cct gca ttc gcc tog gcg 432 Pro Ala Leu Gln Pro Thr Glin Gly Ala Met Pro Ala Phe Ala Ser Ala 13 O 135 14 O titt Caa cqc cqt gcg ggt ggc gtg Ctg gta gcc agc cat ttg cag agc 48O Phe Glin Arg Arg Ala Gly Gly Val Lieu Val Ala Ser His Lieu. Glin Ser 145 150 155 160 titt ctd gag gtg agc tat cqc gtc ct c cqt cat citc gcc caa cc.g 525 Phe Lieu. Glu Val Ser Tyr Arg Val Lieu. Arg His Lieu Ala Glin Pro 1.65 17O 17s

<210s, SEQ ID NO 28 &211s LENGTH: 175 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Synthetic polypeptide

<4 OOs, SEQUENCE: 28 Met Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Glin Ser Phe Leu Lieu. 1. 5 1O 15 Lys Cys Lieu. Glu Glin Val Arg Lys Ile Glin Gly Asp Gly Ala Ala Lieu. 2O 25 3O Glin Glu Lys Lieu. Cys Ala Thr Tyr Lys Lieu. Cys His Pro Glu Glu Lieu. 35 4 O 45 Val Lieu. Lieu. Gly His Ser Lieu. Gly Ile Pro Trp Ala Pro Lieu. Ser Ser SO 55 6 O Cys Pro Ser Glin Ala Lieu. Glin Lieu Ala Gly Cys Lieu. Ser Glin Lieu. His 65 70 7s 8O Ser Gly Lieu. Phe Lieu. Tyr Glin Gly Lieu. Lieu. Glin Ala Lieu. Glu Gly Ile 85 90 95 Ser Pro Glu Lieu. Gly Pro Thr Lieu. Asp Thir Lieu. Glin Lieu. Asp Val Ala 1OO 105 11 O Asp Phe Ala Thr Thr Ile Trp Glin Gln Met Glu Glu Lieu. Gly Met Ala 115 12 O 125 Pro Ala Leu Gln Pro Thr Glin Gly Ala Met Pro Ala Phe Ala Ser Ala 13 O 135 14 O Phe Glin Arg Arg Ala Gly Gly Val Lieu Val Ala Ser His Lieu. Glin Ser 145 150 155 160 Phe Lieu. Glu Val Ser Tyr Arg Val Lieu. Arg His Lieu Ala Glin Pro 1.65 17O 17s

<210s, SEQ ID NO 29

US 8,455,218 B2 55 56 - Continued gactittgatg gcaacggcaa gatcqatctg acctacacct tcc tic acctic gogotacic cag 24 O agcaccatga acaaac atgg Catct cqggg ttcagc.cagt t caac accca gcagaaagca 3OO Caggcc.gcac teccatgca atcctgggcg gatgttgcca acgtgacctt taccgaaaag 360 gctt CC9gcg gtgacggc.ca catgacgttc ggtaactaca gcagtggcca ggacggcgc.c 42O gcggcCtt.cg Cttacctgcc cqgtaccggit gcaggctacg acggcacct C gtggtacctg 48O acaaacaa.ca gct acacgcc gaacaagacic ccggacctga acaactatgg ccggcagacic 54 O Ctgacccacg aaatcggc.ca caccctgggc ctggct Cacc Ctggcgact a caacgc.cggg 6OO aacggcaa.cc cacct at aa cacgcaa.cc tatggaCagg acacgcgtgg ttatagcct c 660 atgagttact ggagcgagag caacaccaac Cagaact tca gcaaaggcgg C9tcgaagct 72 O tacgct tccg gcc.cgctgat cacgacatt gcc.gc.gatcc agaagcticta C9gtgccaac 78O

Ctcagc accc gcgc.cacgga caccacctac gggttcaact C caac accgg gcgtgattt C 84 O Ctcagcgc.ca C9tcCaacgc cgacaagctg gtgttct cqg tatgggacgg togcggcaac 9 OO gacaccctgg acttct cogg titt cacccag aaccagaaga t caacct cac ggccacct cq 96.O ttct Ctgatg tdggcggcct ggtgggcaac gtgtc. catcg cca agggcgt. Caccatcgag 1 O2O aacg.cgttcg gcggcgcggg caacgacctg attattggta accaagttgc Caacaccatc 108 O aagggcgggg ccggcaacga cct catctac ggcggcggcg gtgcggacca actgtggggc 114 O ggcgcgggca gcgata catt cqtgtacggit gcc agttc.cg act coaa.gcc aggggctgcg 12 OO gatalagat.ct tcgactitcac gtc.cggttcg gacaagat.cg acctgtctgg tat caccalag 126 O ggtgcggg.cg tacct tcgt Caacgc.ctitt accgggcatg ccggcgatgc tigtactgagc 132O tatgcct cqg gtacca acct gggcaccittg gcc.gtggact titt Cogggca C9gcgtggcg 1380 gattitcct cq t caccaccgt to Caggcg gctgc.ca.gtg a catcgtagc C 1431

What is claimed is: 9. The method of claim 1, wherein the Pseudomonad host 1. A method comprising producing a soluble G-CSF pro cell is a Pseudomonas host cell. tein in a Pseudomonadhost cell, wherein at least 99% of the 40 10. The method of claim 9, wherein the Pseudomonas host G-CSF protein comprises an N-terminal methionine, wherein cell is a Pseudomonas fluorescens host cell. the Pseudomonadhost cell has a mutation in at least one gene 11. The method of claim 1, wherein the G-CSF protein is encoding at least one protease, wherein the mutation results in human G-CSF protein. the inactivation of the at least one protease, and wherein the at 12. The method of claim 1, further comprising determining least one protease inactivated is PrtB; both PrtB and AprA: 45 the yield of the G-CSF protein, wherein the yield of the both the serine peptidase encoded by SEQID NO: 29 and the S41 protease encoded by SEQID NO:30; an HtpX protease G-CSF protein is about 0.1 g/L to about 10 g/L. encoded by SEQ ID NO: 31; or both the serine peptidase 13. The method of claim 1, further comprising determining encoded by SEQID NO: 29 and the Lon protease encoded by the activity of the G-CSF protein. SEQID NO:32. 14. The method of claim 13, wherein said activity is deter 2. The method of claim 1, wherein the producing comprises 50 mined by binding recombinant G-CSF receptor. expressing the G-CSF protein from an expression construct. 15. A method comprising producing a soluble G-CSF pro 3. The method of claim 2, wherein the expression construct tein in a Pseudomonadhost cell, wherein at least 99% of the is in a plasmid. G-CSF protein comprises an N-terminal methionine, wherein 4. The method of claim 2, wherein the expression construct refolding the Met-G-CSF protein is not required, and wherein comprises a sequence encoding the G-CSF protein fused to a 55 the yield of soluble Met-G-CSF protein is about 0.1 g/L to secretion leader. about 10 g/L. 5. The method of claim 4, wherein the secretion signal 16. The method of claim 15, wherein the producing com directs transfer of the G-CSF protein to the periplasm of the prises expressing the G-CSF protein from an expression con Pseudomonad host cell. Struct. 6. The method of claim 4, wherein the secretion signal is 60 17. The method of claim 16, wherein the expression con cleaved from the G-CSF protein in the Pseudomonad host struct is a plasmid. cell. 18. The method of claim 16, wherein the expression con 7. The method of claim 4, wherein the secretion leader struct comprises a sequence encoding G-CSF protein fused to protein sequence comprises any one of SEQID NOs: 8-26. a secretion leader. 8. The method of claim 1, wherein the mutation that results 65 19. The method of claim 18, wherein the secretion leader in the inactivation of the at least one protease is a complete directs transfer of the G-CSF protein to the periplasm of the deletion. Pseudomonad host cell. US 8,455,218 B2 57 58 20. The method of claim 18, wherein the secretion leader is cleaved from the G-CSF protein in the Pseudomonad host cell. 21. The method of claim 18, wherein the secretion leader comprises any one of SEQID NOs: 8-26. 5 22. The method of claim 15, wherein the Pseudomonad host cell is a Pseudomonas host cell. 23. The method of claim 22, wherein the Pseudomonas host cell is a Pseudomonas fluorescens host cell. 24. The method of claim 15, wherein the G-CSF protein is 10 human G-CSF protein. 25. The method of claim 15, further comprising determin ing the activity of the G-CSF protein. 26. The method of claim 25, wherein the activity is deter mined by binding recombinant G-CSF receptor. 15 k k k k k