US0093.94571B2

(12) United States Patent (10) Patent No.: US 9,394,571 B2 Ramseier et al. (45) Date of Patent: Jul.19, 2016

(54) METHOD FOR RAPIDLY SCREENING 5,043,430 A 8, 1991 Yoshikawa MICROBAL, HOSTS TO DENTIFY CERTAIN 5,055,294 A 19 1991 Gilroy 1 STRANS WITH IMPROVEDYELD AND/OR 392. A 192: Erystal QUALITY IN THE EXPRESSION OF 5,085,862. A 2, 1992 Kleinet al. HETEROLOGOUS PROTENS 5,128,130 A 7/1992 Gilroy et al. 5,151,350 A 9, 1992 Colbert et al. (75) Inventors: Thomas M. Ramseier, Carmel, IN (US); 3.3% A 3. 3: St. Russel J. Coleman, San Diego, CA 5,169,772 A 12/1992 ZimmermanCOX et al. (US); Jane C. Schneider, San Diego, 5,173,616 A 12/1992 Hinooka CA (US); Charles D. Hershberger, 5,232,840 A 8, 1993 Olins Fremont, CA (US); Diane M. Retallack, 5,264.365 A 1 1/1993 Georgiou et al. Poway, CA (US); Charles H. Squires, 5,281,532 A 1/1994 Rammler et al. Poway, CA (US) 5,292,507 A 3, 1994 Charley s 5,292,658 A 3, 1994 Cormier et al. 5,348,867 A 9/1994 Georgiou et al. (73) Assignee: PFENEX INC., San Diego, CA (US) 5,399,684. A 3/1995 Davie et al. 5,418, 155 A 5/1995 Cormier et al. (*) Notice: Subject to any disclaimer, the term of this 5,441,934 A 8/1995 Krapcho et al. patent is extended or adjusted under 35 (Continued) U.S.C. 154(b) by 475 days. FOREIGN PATENT DOCUMENTS (21) Appl. No.: 12/109,554 EP O121352 10, 1984 (22) Filed: Apr. 25, 2008 EP O1551.89 9, 1985 (Continued) (65) Prior Publication Data OTHER PUBLICATIONS US 2008/O26907OA1 Oct. 30, 2008 Wu et al., 2002, Cell-biological applications of transfected-cell Related U.S. Application Data microarrays, Trends in Cell Biology, 12(10): 485-488.* Baneyx. F., and G. Georgiou, "Construction and Characterization of (60) signal application No. 60/914.361, filed on Apr. Escherichia coli Strains Deficient in Multiple Secreted Protease: s Protease III Degrades High-Molecular-Weight Substrates InVivo..”.J. Bacteriol., Apr. 1991, pp. 2696-2703, vol. 173, No. 8. (51) Int. Cl. Thomas, J.G., et al., Molecular Chaperones, Folding Catalysts, and GOIN33/554 (2006.01) the Recovery of Active Recombinant Proteins from E. coli To Fold CI2N L/20 (2006.01) or to Refold, Applied Biochemistry and Biotechnology, 1997, pp. CI2N IS/00 (2006.01) 197-238, vol. 66. CI2P 2L/04 (2006.01) Wall, G.J., and Pluckthun, A., “Effects of Overexpressing Folding CI2P2/06 (2006.01) Modulators on the in vivo Folding of Heterologous Proteins in C7H 2L/04 (2006.01) Escherichia coli," Current Opinion in Biotechnology, Jan. 1, 1995, CI2O I/68 (2006.01) pp. 507-516, vol. 6, London, GB |XP02092905). CI2N 15/78 (2006.01) (Continued) (52) U.S. Cl. CPC CI2O I/689 (2013.01); C12N 1/20 (2013.01); Primary Examiner —Yong Pak CI2N 15/78 (2013.01) (74) Attorney, Agent, or Firm — Wilson Sonsini Goodrich & (58) Field of Classification Search Rosati CPC ...... C12N 1/20; C12N 15/78; C12Q 1 Fé89 (57) ABSTRACT See application file for complete search history. The present invention provides an array for rapidly identify (56) References Cited ing a host cell population capable of producing a heterolo gous protein with improved yield and/or quality. The array U.S. PATENT DOCUMENTS comprises one or more host cell populations that have been genetically modified to increase the expression of one or more 3,844,893 A 10, 1974 Hitzman target genes involved in protein production, decrease the 3,878,093 A 4, 1975 Kanani et al. 4,169,010 A 9, 1979 Marwill expression of one or more target genes involved in protein 4,432,895 A 2f1984 Tamowski degradation, or both. One or more of the strains in the array 4,511,503 A 4, 1985 Olson et al. may express the heterologous protein of interest in a peri 4,551,433 A 11, 1985 DeBoer plasm compartment or may secrete the heterologous protein 4,595,658 A 6, 1986 Zinder et al. extracellularly through an outer cell wall. The strain arrays 4,637,980 A 1, 1987 Auerbach et al. are useful for Screening for improved expression of any pro 4,680,264 A 7, 1987 Puhler et al. tein of interest including therapeutic proteins, hormones, 4,695.455 A 9, 1987 Bames et al. 4,755.465 A 7/1988 Gray et al. growth factors, extracellular receptors or ligands, proteases, 4,861,595 A 8, 1989 Bames et al. kinases, blood proteins, chemokines, cytokines, antibodies 4,888,274 A 12/1989 Radding et al. and the like. 4,963,495 A 10/1990 Chang et al. 5,023,171 A 6, 1991 Ho et al. 13 Claims, 3 Drawing Sheets US 9,394,571 B2 Page 2

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US 9,394,571 B2 1. 2 METHOD FOR RAPIDLY SCREENING Publication No. WO 89/10971; U.S. Pat. No. 6,156,552; U.S. MICROBAL, HOSTS TO DENTIFY CERTAIN Pat. Nos. 6,495,357; 6,509, 181; 6,524,827; 6,528,298; 6,558, STRANS WITH IMPROVEDYELD AND/OR 939; 6,608,018; 6,617,143: U.S. Pat. Nos. 5,595,898; 5,698, QUALITY IN THE EXPRESSION OF 435; and 6,204,023; U.S. Pat. No. 6,258,560; PCT Publica HETEROLOGOUS PROTEINS 5 tion Nos. WO 01/21662, WO 02/068660 and U.S. Application Publication 2003/0044906: U.S. Pat. No. 5,641, CROSS-REFERENCE TO RELATED 671; and European Patent No. EP 0121352. APPLICATIONS Heterologous protein production often leads to the forma tion of insoluble or improperly folded proteins, which are This application claims the benefit of U.S. Provisional difficult to recover and may be inactive. Furthermore, the Application No. 60/914,361, filed Apr. 27, 2007, which is presence of specific host cell proteases may degrade the pro hereby incorporated in its entirety by reference herein. tein of interest and thus reduce the final yield. There is no single factor that will improve the production of all heterolo REFERENCE TO ASEQUENCE LISTING gous proteins. As a result, there is a need in the art for iden SUBMITTED ASATEXT FILE VIA EFS-WEB 15 tifying improved large-scale expression systems capable of secreting and properly processing recombinant polypeptides The official copy of the sequence listing is Submitted con to produce transgenic proteins in properly processed form. currently with the specification as a text file via EFS-Web, in compliance with the American Standard Code for Informa SUMMARY OF THE INVENTION tion Interchange (ASCII), with a file name of 20 3.42528 SequenceListing..txt, a creation date of Apr. 21, 2008 The present invention provides compositions and methods and a size of 216 KB. The sequence listing filed via EFS-Web for rapidly identifying a host cell population capable of pro is part of the specification and is herein incorporated by ducing at least one heterologous polypeptide according to a reference in its entirety. desired specification with improved yield and/or quality. The 25 compositions comprise two or more host cell populations that FIELD OF THE INVENTION have been genetically modified to increase the expression of one or more target genes involved in protein production, This invention is in the field of protein production, particu decrease the expression of one or more target genes involved larly to identifying optimal host cells for large-scale produc in protein degradation, express a heterologous gene that tion of properly processed heterologous proteins. 30 affects the protein product, or a combination. The ability to express a polypeptide of interest in a variety of modified host BACKGROUND OF THE INVENTION cells provides a rapid and efficient means for determining an optimal host cell for the polypeptide of interest. The desired More than 150 recombinantly produced proteins and specification will vary depending on the polypeptide of inter polypeptides have been approved by the U.S. Food and Drug 35 est, but includes yield, quality, activity, and the like. Administration (FDA) for use as biotechnology drugs and It is recognized that the host cell populations may be modi vaccines, with another 370 in clinical trials. Unlike small fied to express many combinations of nucleic acid sequences molecule therapeutics that are produced through chemical that affect the expression levels of endogenous sequences synthesis, proteins and polypeptides are most efficiently pro and/or exogenous sequences that facilitate the expression of duced in living cells. However, current methods of production 40 the polypeptide of interest. In one embodiment, two or more of recombinant proteins in bacteria often produce improperly of the host cell populations has been genetically modified to folded, aggregated or inactive proteins, and many types of increase the expression of one or more target genes involved proteins require secondary modifications that are inefficiently in one or more of the proper expression, processing, and/or achieved using known methods. translocation of a heterologous protein of interest. In another Numerous attempts have been developed to increase pro 45 embodiment, the target gene is a protein folding modulator. In duction of properly folded proteins in recombinant systems. another embodiment, the protein folding modulator is For example, investigators have changed fermentation con selected from the list in Table 1. ditions (Schein (1989) Bio/Technology, 7:1141-1149), varied In another embodiment, one or more of the host cell popu promoter strength, or used overexpressed chaperone proteins lations has been genetically modified to decrease the expres (Hockney (1994) Trends Biotechnol. 12:456-463), which can 50 sion of one or more target genes involved in proteolytic deg help prevent the formation of inclusion bodies. radation. In another embodiment, the target gene is a Strategies have been developed to excrete proteins from the protease. In another embodiment, the protease is selected cell into the supernatant. For example, U.S. Pat. No. 5,348, from the list in Table 2. 867; U.S. Pat. No. 6,329, 172: PCT Publication No. WO In one embodiment, nucleotide sequences encoding the 96/17943; PCT Publication No. WO 02/40696; and U.S. 55 proteins of interest are operably linked to a Pfluorescens Sec Application Publication 2003/0013150. Other strategies for system secretion signal as described herein. One or more of increased expression are directed to targeting the protein to the strains in the array may express the heterologous protein the periplasm. Some investigations focus on non-Sec type of interest in a periplasm compartment. In certain embodi secretion (see for e.g. PCT Publication No. WO 03/079007: ments, one or more strains may also secrete the heterologous U.S. Publication No. 2003/0180937; U.S. Publication No. 60 protein extracellularly through an outer cell wall. 2003/0064.435; and, PCT Publication No. WO 00/59537). Host cells include eukaryotic cells, including yeast cells, However, the majority of research has focused on the secre insect cells, mammalian cells, plant cells, etc., and prokary tion of exogenous proteins with a Sec-type secretion system. otic cells, including bacterial cells such as Pfluorescens, E. A number of secretion signals have been described for use coli, and the like. in expressing recombinant polypeptides or proteins. See, for 65 As indicated, the library of host cell populations can be example, U.S. Pat. No. 5,914,254; U.S. Pat. No. 4.963,495; rapidly screened to identify certain strain(s) with improved European Patent No. 0 177343; U.S. Pat. No. 5,082,783; PCT yield and/or quality of heterologously expressed protein. The US 9,394,571 B2 3 4 strain arrays are useful for screening for improved expression affects heterologous protein production in a host cell. Target of any protein of interest, including therapeutic proteins, hor genes that affect heterologous protein production include mones, a growth factors, extracellular receptors or ligands, genes encoding proteins that modulate expression, activity, proteases, kinases, blood proteins, chemokines, cytokines, solubility, translocation, proteolytic degradation and/or antibodies and the like. cleavage of the heterologous protein. For example, a target gene may encode at least one of a host cell protease, a protein BRIEF DESCRIPTION OF THE FIGURES folding modulator, a transcription factor, a translation factor, a secretion modulator, or any other protein involved in the FIG. 1A depicts plasmid plCW1261-2 used for engineer proper transcription, translation, processing, and/or translo ing genomic deletion in Pfluorescens. FIG.1B is a schematic 10 cation of a heterologous protein of interest. A “target protein’ drawing of the constructions of a gene X deletion. refers to the protein or polypeptide resulting from expression FIG. 2 is a Western blot analysis of soluble cells fractions prepared at 0 and 24 hours post-induction (IO and I24, respec of the target gene. Expression and/or activity of a target gene tively) in AprC1, AdegP2, ALa2 and the grpEdnaKJ co-ex or genes is increased or decreased, depending on the function pression strains (Example 6). The top arrows point to the fully 15 of the target gene or protein. For example, expression of one assembled monoclonal antibody in the co-expressed strains or more host cell proteases may be decreased, whereas but not in the control (DC440). r-recombinant; expression of one or more protein folding modulators may be n-r nonrecombinant. increased. The arrays described herein are useful for rapidly identi DETAILED DESCRIPTION fying an optimal host cell for production of a heterologous protein or peptide of interest. Heterologous protein produc The present inventions now will be described more fully tion often leads to the formation of insoluble or improperly hereinafter with reference to the accompanying drawings, in folded proteins, which are difficult to recover and may be which some, but not all embodiments of the invention are inactive. Furthermore, the presence of specific host cell pro shown. Indeed, these inventions may be embodied in many 25 teases may degrade the protein of interest and thus reduce the different forms and should not be construed as limited to the final yield. There is no single host cell population that will embodiments set forth herein; rather, these embodiments are optimally produce all polypeptides or proteins of interest. provided so that this disclosure will satisfy applicable legal Thus, using the compositions and methods of the invention, requirements. an optimal host cell can be rapidly and efficiently identified Many modifications and other embodiments of the inven 30 from the library of modified cell populations. The optimal tions set forth herein will come to mind to one skilled in the art host strain can then be used to produce Sufficient amounts of to which these inventions pertain having the benefit of the the protein of interest or for commercial production. Like teachings presented in the foregoing descriptions and the wise, a host strain can be modified for expression of the associated drawings. protein of interest based on the optimal host strain. Therefore, it is to be understood that the inventions are not 35 In one embodiment, the method includes obtaining an to be limited to the specific embodiments disclosed and that array comprising at least a first and a second population of P modifications and other embodiments are intended to be fluorescens cells, wherein each population is selected from included within the scope of the invention. Although specific the group consisting of (i) a population of Pfluorescens cells terms are employed herein, they are used in a generic and that has been genetically modified to reduce the expression of descriptive sense only and not for purposes of limitation. 40 at least target gene involved in protein degradation; (ii) a Overview population of Pfluorescens cells that has been genetically Compositions and methods for identifying an optimal host modified to increase the expression of at least one target gene strain, e.g., a Pseudomonas fluorescens host strain, for pro involved in protein production; and, (iii) a population of P ducing high levels of properly processed heterologous fluorescens cells that has been genetically modified to reduce polypeptides in a host cell are provided. In particular, a library 45 the expression of at least one target gene involved in protein (or "array') of hoststrains is provided, wherein each strain (or degradation and to increase the expression of at least one “population of host cells') in the library has been genetically target gene involved in protein production; introducing into at modified to modulate the expression of one or more target least one cell of each population an expression construct genes in the host cell. An “optimal host strain” can be iden comprising at least one gene encoding at least one heterolo tified or selected based on the quantity, quality, and/or loca 50 gous protein of interest; maintaining said cells under condi tion of the expressed protein of interest compared to other tions sufficient for the expression of said protein of interest in populations of phenotypically distinct host cells in the array. at least one population of cells; and selecting the optimal Thus, an optimal host strain is the strain that produces the population of cells in which the heterologous protein of inter polypeptide of interest according to a desired specification. est is produced; wherein each population in the array is non While the desired specification will vary depending on the 55 identical and wherein each population is physically separate polypeptide being produced, the specification includes the from one from another; wherein the heterologous protein of quality and/or quantity of protein, whether the protein is interest exhibits one or more of improved expression, sequestered or secreted, protein folding, and the like. improved activity, improved solubility, improved transloca “Heterologous.” “heterologously expressed, or “recombi tion, or reduced proteolytic degradation or cleavage in the nant generally refers to a gene or protein that is not endog 60 optimal population of cells compared to other populations in enous to the host cell or is not endogenous to the location in the array. the native genome in which it is present, and has been added The array may further comprise a population of host cells to the cell by infection, transfection, microinjection, elec (e.g., Pfluorescens host cells) that has not been genetically troporation, microprojection, or the like. modified to alter the expression of a host cell protease or a One or more of the host cell populations in the array is 65 protein folding modulator. This population may be a wild modified to modulate the expression of one or more target type strain, or may be a strain that has been genetically modi genes in the host cell. By “target gene' is intended a gene that fied to alter the expression of or more genes not involved in US 9,394,571 B2 5 6 protein production, processing, or translocation (e.g., may be arrays may be created and/or screened using a spotter device genetically modified to express, for example, a selectable (e.g., automated robotic devices) as known in the art. marker gene). Target Genes In one embodiment, each population of Pfluorescens host The strain array of the present invention comprises a plu cells is phenotypically distinct (i.e., "non-identical') one 5 rality of phenotypically and genotypically distinct host cell from another. By “phenotypically distinct” is intended that populations, wherein each population in the array has been each population produces a measurably different amount of genetically modified to modulate the expression of one or one or more target proteins. In this embodiment, each strain more target genes in the host cell. By “target gene' is intended has been genetically modified to alter the expression of one or a gene that affects heterologous protein production in a host 10 cell. A target gene may encode a host cell protease or an more different target genes. Where the expression of more endogenous or exogenous protein folding modulator, tran than one target gene is modulated in a population of host cells, Scription factor, translation factor, secretion modulator, or then the combination of target genes is phenotypically dis any other gene involved in the proper expression, processing, tinct from other populations in the library. An array compris and/or translocation of a heterologous protein of interest. A ing a plurality of phenotypically distinct populations of host 15 “target protein refers to the protein or polypeptide resulting cells according to the present invention is one that provides a from expression of the target gene. Expression and/or activity diverse population from which to select one or more strains of a target gene or genes is increased or decreased, depending useful for producing a heterologous protein or peptide of on the function of the target gene or protein. A target gene can interest. It will be understood by one of skill in the art that be endogenous to the host cell, or can be a gene that is Such an array may also comprise replicates (e.g., duplicates, heterologously expressed in each of the host cell populations triplicates, etc.) of any one or more populations of host cells. in the array. Arrays In one embodiment, the target gene or genes is at least one Provided herein is an array of host cell populations (i.e. protein folding modulator, putative protein folding modula “strain array') which can be rapidly screened to identify tor, or a cofactor or subunit of a folding modulator. In some certain strain(s) with improved yield and/or quality of heter 25 embodiments, the target gene or genes can be selected from a ologous protein. As used herein, the term “strain array refers chaperone protein, a foldase, a peptidyl prolyl isomerase and to a plurality of addressed or addressable locations (e.g., a disulfide bond isomerase. In some embodiments, the target wells, such as deep well or microwells). The location of each gene or genes can be selected from htpG, cbp.A, dna, dnaK of the microwells or groups of and flkbP. Exemplary protein folding modulators from Pfluo microwells in the array is typically known, so as to allow for 30 rescens are listed in Table 1. identification of the optimal host cell for expression of the In other embodiments, the target gene comprises at least heterologous protein of interest. one putative protease, a protease-like protein, or a cofactor or The strain array comprises a plurality of phenotypically Subunit of a protease. For example, the target gene or genes distinct host strains. The arrays may be low-density arrays or can be a serine, threonine, cysteine, aspartic or metallopepti high-density arrays and may contain about 2 or more, about 4 35 dase. In one embodiment, the target gene or genes can be or more, about 8 or more, about 12 or more, about 16 or more, selected from hslV, hslU, clp A, clp3 and clpX. The target about 20 or more, about 24 or more, about 32 or more, about gene can also be a cofactor of a protease. Exemplary proteases 40 or more, about 48 or more, about 64 or more, about 72 or from Pfluorescens are listed in Table 2. Proteases from a more, about 80 or more, about 96 or more, about 192 or more, variety of organisms can be found in the MEROPS Peptidase about 384 or more host cell populations. 40 Database maintained by the Welcome Trust Sanger Institute, The host cell populations of the invention can be main Cambridge, UK (Rawlings et al., 2006, Nucleic Acids tained and/or screened in a multi-well or deep well vessel. Research 34 (Database issue): D2702). The vessel may contain any desired number of wells, how Protein Folding Modulators ever, a miniaturized cell culture microarray platform is useful Another major obstacle in the production of heterologous for screening each population of host cells individually and 45 proteins in host cells is that the cell often is not adequately simultaneously using minimal reagents and a relatively small equipped to produce either soluble or active protein. While number of cells. A typical multi-well, microtiter vessel useful the primary structure of a protein is defined by its amino acid in this assay is a multi-well plate including, without limita sequence, the secondary structure is defined by the presence tion, 10-well plates, 28-well plates, 96-well plates, 384-well of alpha helices or beta sheets, and the ternary structure by plates, and plates having greater than 384 wells. Alternatively, 50 covalent bonds between adjacent protein stretches, such as an array of tubes, holders, cartridges, minitubes, microfuge disulfide bonds. When expressing heterologous proteins, par tubes, cryovials, square well plates tubes, plates, slants, or ticularly in large-scale production, the secondary and tertiary culture flasks may also be used, depending on the Volume structure of the protein itself is of critical importance. Any desired. significant change in proteinstructure can yield a functionally The vessel may be made of any material suitable for cul 55 inactive molecule, or a protein with significantly reduced turing and/or screening a host cell of interest, e.g., Pseudomo biological activity. In many cases, a host cell expresses pro nas. For example, the vessel can be a material that can be tein folding modulators (PFMs) that are necessary for proper easily Sterilized such as plastic or other artificial polymer production of active heterologous protein. However, at the material. So long as the material is biocompatible. Any num high levels of expression generally required to produce ber of materials can be used, including, but not limited to, 60 usable, economically satisfactory biotechnology products, a polystyrene; polypropylene; polyvinyl compounds (e.g. cell often cannot produce enough native protein folding polyvinylchloride); polycarbonate (PVC); polytetrafluoroet modulator or modulators to process the heterologously-ex hylene (PTFE); polyglycolic acid (PGA); cellulose; glass, pressed protein. fluoropolymers, fluorinated ethylene propylene, polyvi In certain expression systems, overproduction of heterolo nylidene, polydimethylsiloxane, silicon, and the like. 65 gous proteins can be accompanied by their misfolding and Automated transformation of cells and automated colony segregation into insoluble aggregates. In bacterial cells these pickers will facilitate rapid screening of desired cells. The aggregates are known as inclusion bodies. In E. coli, the US 9,394,571 B2 7 8 network of folding modulators/chaperones includes the intra-protein disulfide bonds. Any protein that has more than Hsp70 family. The major Hsp70 chaperone, DnaK, efficiently two cysteines is at risk of forming disulfidebonds between the prevents protein aggregation and Supports the refolding of wrong residues. The disulfide bond formation family consists damaged proteins. The incorporation of heat shock proteins of the Dsb proteins which catalyze the formation of disulfide into protein aggregates can facilitate disaggregation. How bonds in the non-reducing environment of the periplasm. ever, proteins processed to inclusion bodies can, in certain When a periplasmic polypeptide misfolds disulfide bond cases, be recovered through additional processing of the isomerase, DsbC is capable of rearranging the disulfide bonds insoluble fraction. Proteins found in inclusion bodies typi and allowing the protein to reform with the correct linkages. cally have to be purified through multiple steps, including 10 The proline residue is unique among amino acids in that the denaturation and renaturation. Typical renaturation processes peptidyl bond immediately preceding it can adopt either a cis for inclusion body targeted proteins involve attempts to dis Solve the aggregate in concentrated denaturant and Subse or trans conformation. For all other amino acids this is not quent removal of the denaturant by dilution. Aggregates are favored due to steric hindrance. Peptidyl-prolyl cis-trans frequently formed again in this stage. The additional process isomerases (PPIases) catalyze the conversion of this bond ing adds cost, there is no guarantee that the in vitro refolding 15 from one form to the other. This isomerization may aid in will yield biologically active product, and the recovered pro protein folding, refolding, assembly of Subunits and traffick teins can include large amounts of fragment impurities. ing in the cell (Dolinski, et. al. 1997). The recent realization that in vivo protein folding is In addition to the general chaperones which seem to inter assisted by molecular chaperones, which promote the proper act with proteins in a non-specific manner, there are also isomerization and cellular targeting of other polypeptides by chaperones which aid in the folding of specific targets. These transiently interacting with folding intermediates, and by fol protein-specific chaperones form complexes with their tar dases, which accelerate rate-limiting steps along the folding gets, preventing aggregation and degradation and allowing pathway, has provided additional approaches to combat the time for them to assemble into multi-subunit structures. The problem of inclusion body formation (see for e.g. Thomas JG 25 PapD chaperone is one well known example of this type et al. (1997) Appl Biochem Biotechnol 66:197-238). (Lombardo et al. 1997). In certain cases, the overexpression of chaperones has been Folding modulators also include, for example, HSP70 pro found to increase the soluble yields of aggregation-prone teins, HSP110/SSE proteins, HSP40 (DNAJ-related) pro proteins (see Baneyx, F. (1999) Curr. Opin. Biotech. 10:411 30 teins, GRPE-like proteins, HSP90 proteins, CPN60 and 421 and references therein). The beneficial effect associated CPN10 proteins, Cytosolic chaperoning, HSP 100 proteins, with an increase in the intracellular concentration of these Small HSPs, Calnexin and calreticulin, PDI and thioredoxin chaperones appears highly dependent on the nature of the related proteins, Peptidyl-prolyl isomerases, Cyclophilin overproduced protein, and may not require overexpression of PPIases, FK-506 binding proteins, Parvulin PPIases, Indi the same protein folding modulator(s) for all heterologous 35 vidual chaperoning, Protein specific chaperones, or intramo proteins. lecular chaperones. Folding modulators are generally Protein folding modulators, including chaperones, disul described in "Guidebook to Molecular Chaperones and Pro fide bond isomerases, and peptidyl-prolyl cis-trans tein-Folding Catalysts’ (1997) ed. M. Gething, Melbourne isomerases (PPIases) area class of proteins present in all cells University, Australia. which aid in the folding, unfolding and degradation of 40 The best characterized molecular chaperones in the cyto nascent polypeptides. plasm of E. coli are the ATP-dependent DnaK-DnaJ-GrpE Chaperones act by binding to nascent polypeptides, stabi and GroEL-GroES systems. Based on in vitro studies and lizing them and allowing them to fold properly. Proteins homology considerations, a number of additional cytoplas possess both hydrophobic and hydrophilic residues, the 45 mic proteins have been proposed to function as molecular former are usually exposed on the surface while the latter are chaperones in E. coli. These include Clpb, HtpG and IbpA/B, buried within the structure where they interact with other which, like DnaK-DnaJ-GrpE and GroEL-GroES, are heat hydrophilic residues rather than the water which surrounds shock proteins (Hsps) belonging to the stress regulon. The the molecule. However in folding polypeptide chains, the trans conformation of X-Pro bonds is energetically favored in hydrophilic residues are often exposed for some period of 50 nascent protein chains; however, approximately 5% of all time as the protein exists in a partially folded or misfolded prolyl peptide bonds are found in a cis conformation in native state. It is during this time when the forming polypeptides can proteins. The trans to cis isomerization of X-Pro bonds is rate become permanently misfolded or interact with other mis limiting in the folding of many polypeptides and is catalyzed folded proteins and form large aggregates or inclusion bodies 55 in vivo by peptidyl prolyl cis/trans isomerases (PPIases). within the cell. Chaperones generally act by binding to the Three cytoplasmic PPIases, SlyD, Slp A and trigger factor hydrophobic regions of the partially folded chains and pre (TF), have been identified to date in E. coli. TF, a 48 kDa venting them from misfolding completely or aggregating protein associated with 50S ribosomal subunits that has been with other proteins. Chaperones can even bind to proteins in postulated to cooperate with chaperones in E. coli to guaran inclusion bodies and allow them to disaggregate (Ranson et. 60 tee proper folding of newly synthesized proteins. At least five al. 1998). The GroES/EL, DnaKJ, Clp, Hsp90 and SecB proteins (thioredoxins 1 and 2, and glutaredoxins 1, 2 and 3. families of folding modulators are all examples of proteins the products of the trXA, trXc, grXA, grxB and grXC genes, with chaperone like activity. respectively) are involved in the reduction of disulfide bridges Another important type of folding modulator is the disul 65 that transiently arise in cytoplasmic enzymes. Thus, target fide bond isomerases. These proteins catalyze a very specific genes can be disulfide bond forming proteins or chaperones set of reactions to help folding polypeptides form the proper that allow proper disulfide bond formation. US 9,394,571 B2

TABLE 1 A fluorescens Strain MB214 protein folding modulators

ORFID GENE FUNCTION FAMILY LOCATION GroESEL RXFO2095. groES Chaperone Hsp10 Cytoplasmic RXFO6767.1: groEL Chaperone Hsp60 Cytoplasmic Rxf)2O90 RXFO1748. ibp A Small heat-shock protein (sESP) Ibp A HS2O Cytoplasmic PA3126; Acts as a holder for GroESL folding RXFO3385. hscE Chaperone protein hsch HS2O Cytoplasmic Hsp70 (DnaK/J) RXFOS399. dnaK Chaperone Hsp70 Periplasmic RXFO69S4. dnaK Chaperone Hsp70 Cytoplasmic RXFO3376. hscA Chaperone Hsp70 Cytoplasmic RXFO3987.2 cbp.A Curved dina-binding protein, dinal like activity Hsp40 Cytoplasmic RXFOS4O6.2 dna Chaperone protein dinal Hsp40 Cytoplasmic RXFO3346.2 dna Molecular chaperones (DnaJ family) Hsp40 Non-secretory RXFOS413. grpE heat shock protein GrpE PA4762 GrE Cytoplasmic Hsp100 (Clp/Hsl) RXFO4587. clpA atp-dependent clp protease atp-binding Subunit Hsp100 Cytoplasmic clip A RXFO8347. clpB ClpB protein Hsp100 Cytoplasmic RXFO4654.2 clpX atp-dependent clp protease atp-binding Subunit Hsp100 Cytoplasmic clpX RXFO4663. clpP atp-dependent Clp protease proteolytic subunit MEROPS Cytoplasmic (ec 3.4.21.92) peptidase amily S14 RXFO1957.2 hSIU atp-dependenths protease atp-binding subunit Hsp100 Cytoplasmic hisU RXFO1961.2 hSIV atp-dependenths protease proteolytic subunit MEROPS Cytoplasmic peptidase subfamily T1B Hsp33 RXFO4254.2 yrfI 33 kDa chaperonin (Heat shock protein 33 Hsp33 Cytoplasmic homolog) (HSP33). Hsp90 RXFOS455.2 htpG Chaperone protein htpG Hsp90 Cytoplasmic SecB RXFO2231.1 SecB secretion specific chaperone SecB SecB Non-secretory Disulfide Bond Isomerases RXFO7017.2 disbA disulfide isomerase DSBA oxido- Cytoplasmic reductase RXFO8657.2 disbA disulfide isomerase DSBA oxido- Cytoplasmic dsbC reductase dsbGif fernA RXFO1 OO2.1 disbA disulfide isomerase DSBA oxido- Periplasmic dsbC reductase? Thioredoxin RXFO3307.1 dsbC disulfide isomerase glutaredoxin. Periplasmic Thioredoxin RXFO4890.2 dsbG disulfide isomerase glutaredoxin. Periplasmic Thioredoxin Peptidyl-prolyl cis-trans isomerases RXFO3768.1 ppiA Peptidyl-prolyl cis-trans isomerase A (ec 5.2.1.8) PPIase: Periplasmic cyclophilin type RXFOS345.2 ppiB Peptidyl-prolyl cis-trans isomerase B. PPIase: Cytoplasmic cyclophilin type RXFO6034.2 fkIB Peptidyl-prolyl cis-trans isomerase FklB. PPIase: OuterMembrane FKBP type RXFO6591.1 fkIB, fk506 binding protein Peptidyl-prolyl cis-trans PPIase: Periplasmic fkbP isomerase (EC 5.2.1.8) FKBP type RXFO5753.2 fkIB; Peptidyl-prolyl cis-trans isomerase (ec 5.2.1.8) PPIase: Outer fkbP FKBP type Membrane RXFO1833.2 slyD Peptidyl-prolyl cis-trans isomerase SlyD. PPIase: Non-secretory FKBP type RXFO46SS.2 tig Trigger factor, ppiase (ec 5.2.1.8) PPIase: Cytoplasmic FKBP type US 9,394,571 B2 11 12 TABLE 1-continued A fluorescens Strain MB214 protein folding modulators

ORFID GENE FUNCTION FAMILY LOCATION RXFOS385. yaad Probable FKBP-type 16 kDa peptidyl-prolyl cis PPIase: Non-secretory trans isomerase (EC 5.2.1.8) (PPiase) FKBP type (Rotamase). RXFOO271. Peptidyl-prolyl cis-trans isomerase (ec 5.2.1.8) PPIase: Non-secretory FKBP type pili assembly chaperones (papD like) RXFO6068. Cl Chaperone protein cup pili assembly Periplasmic bap) RXFO5719. ecpD Chaperone protein ecpD pili assembly Signal peptide bap) RXFO34O6.2 ecpD; Chaperone protein ecpD pili assembly Signal peptide cSuC bap) RXFO4296. ecpD; Chaperone protein ecpD pili assembly Periplasmic Cl bap) RXFO4S53. ecpD; Chaperone protein ecpD pili assembly Periplasmic Cl bap) RXFO4554.2 ecpD; Chaperone protein ecpD pili assembly Periplasmic Cl bap) RXFOS31 O.2 ecpD; Chaperone protein ecpD pili assembly Periplasmic Cl bap) RXFOS3O4.1 ecpD; Chaperone protein ecpD pili assembly Periplasmic Cl bap) RXFO5073.1 gltF Gram-negative pili assembly chaperone pili assembly Signal peptide periplasmic function bap)

Protease cell populations and the level of proteolytic degradation can Unwanted degradation of heterologously-expressed pro be used to identify the optimal host cell. In this embodiment, tein presents an obstacle to the efficient use of certain expres 30 the optimal host cell population is that which results in the sion systems. When a cell is modified to produce large quan least amount of heterologous protein degradation. Thus, in tities of a target protein, the cell is placed under stress and one embodiment, lysate from the optimal host cell population often reacts by inducing or Suppressing other proteins. The can be degraded by less than about 50% of the heterologous stress that a host cell undergoes during production of heter 35 protein, less than about 45%, less than about 40%, less than ologous proteins can increase expression of for example, about 35%, less than about 30%, less than about 25%, less specific proteins or cofactors to cause degradation of the than about 20%, less than about 10%, less than about 5%, less overexpressed heterologous protein. The increased expres than about 4%, about 3%, about 2%, about 1%, or less of the sion of compensatory proteins can be counterproductive to protein. the goal of expressing high levels of active, full-length heter 40 Exemplary target protease genes include those proteases ologous protein. Decreased expression or lack of adequate classified as Aminopeptidases; Dipeptidases; Dipeptidyl expression of other proteins can cause misfolding and aggre peptidases and tripeptidyl peptidases; Peptidyl-dipeptidases; gation of the heterologously-expressed protein. While it is Serine-type carboxypeptidases; Metallocarboxypeptidases; known that a cellunder stress will change its profile of protein Cysteine-type carboxypeptidases; Omegapeptidases; Serine expression, not all heterologously expressed proteins will 45 proteinases; Cysteine proteinases; Aspartic proteinases; Met modulate expression of the same proteins in a particular host allo proteinases; or Proteinases of unknown mechanism. cell. Aminopeptidases include cytosol aminopeptidase (leucyl Thus, the optimal host strain, e.g., P. fluorescens host aminopeptidase), membrane alanyl aminopeptidase, cystinyl strain, can be identified using an array comprising a plurality aminopeptidase, tripeptide aminopeptidase, prolyl ami of host cell populations that have been genetically engineered 50 nopeptidase, arginyl aminopeptidase, glutamyl aminopepti to decrease the expression of one or more protease enzymes. dase, X-pro aminopeptidase, bacterial leucylaminopeptidase, In one embodiment, one or more host cell populations is thermophilic aminopeptidase, clostridial aminopeptidase, modified by reducing the expression of inhibiting or remov cytosol alanyl aminopeptidase, lysyl aminopeptidase, X-trp ing at least one protease from the genome. The modification aminopeptidase, tryptophanyl aminopeptidase, methionyl can also be to more than one protease. In a related embodi 55 aminopeptidas, d-stereospecific aminopeptidase, aminopep ment, the cell is modified by reducing the expression of a tidase ey. Dipeptidases include X-his dipeptidase, X-arg protease cofactor or protease protein. In another embodiment, dipeptidase, X-methyl-his dipeptidase, cys-gly dipeptidase, the host cell is modified by inhibition of a promoter for a glu-glu dipeptidase, pro-X dipeptidase, X-pro dipeptidase, protease or related protein, which can be a native promoter. met-X dipeptidase, non-stereospecific dipeptidase, cytosol Alternatively, the gene modification can be to modulate a 60 non-specific dipeptidase, membrane dipeptidase, beta-ala-his protein homologous to the target gene. dipeptidase. Dipeptidyl-peptidases and tripeptidyl peptidases The array comprising the modified host strains can be include dipeptidyl-peptidasei, dipeptidyl-peptidase ii, dipep screened by expressing the heterologous protein(s) of interest tidyl peptidase iii, dipeptidyl-peptidase iv, dipeptidyl-dipep and assessing the quality and/or quantity of protein produc tidase, tripeptidyl-peptidase I, tripeptidyl-peptidase II. Pepti tion as discussed infra. Alternatively, an isolate of the heter 65 dyl-dipeptidases include peptidyl-dipeptidase a and peptidyl ologous protein of interest can be independently incubated dipeptidase b. Serine-type carboxypeptidases include with lysate collected from each of the protease-deficient host lysosomal pro-X carboxypeptidase, serine-type D-ala-D-ala US 9,394,571 B2 13 14 carboxypeptidase, carboxypeptidase C, carboxypeptidase D. factor c, limulus clotting factor, limulus clotting enzyme, Metallocarboxypeptidases include carboxypeptidase a, car omptin, repressor lexa, bacterial leader peptidase I, togavirin, boxypeptidase B, lysine(arginine) carboxypeptidase, gly-X flavirin. Cysteine proteinases include cathepsin B, papain, ficin, chymopapain, asclepain, clostripain, Streptopain, carboxypeptidase, alanine carboxypeptidase, muramoylpen actinide, cathepsin 1, cathepsin H, calpain, cathepsint, gly tapeptide carboxypeptidase, carboxypeptidase h, glutamate cyl, endopeptidase, cancer procoagulant, cathepsin S, picor carboxypeptidase, carboxypeptidase M. muramoyltetrapep nain 3C, picornain 2A, caricain, ananain, stem bromelain, tide carboxypeptidase, Zinc d-ala-d-ala carboxypeptidase, fruit bromelain, legumain, histolysain, interleukin 1-beta carboxypeptidase A2, membrane pro-X carboxypeptidase, converting enzyme. Aspartic proteinases include pepsin A, tubulinyl-tyr carboxypeptidase, carboxypeptidase t. Omega pepsin B, gastricsin, chymosin, cathepsin D. neopenthesin, peptidases include acylaminoacyl-peptidase, peptidyl-glyci 10 renin, retropepsin, pro-opiomelanocortin converting enzyme, namidase, pyroglutamyl-peptidase I, beta-aspartyl-pepti aspergillopepsin I, aspergillopepsin II, penicillopepsin, dase, pyroglutamyl-peptidase II, n-formylmethionyl rhizopuspepsin, endothiapepsin, mucoropepsin, candidapep peptidase, pteroylpoly-gamma-glutamate sin, Saccharopepsin, rhodotorulapepsin, physaropepsin, acro carboxypeptidase, gamma-glu-X carboxypeptidase, acylmu cylindropepsin, polyporopepsin, pycnoporopepsin, Scytali ramoyl-ala peptidase. Serine proteinases include chymot 15 dopepsina, Scytalidopepsin b, Xanthomonapepsin, cathepsin rypsin, chymotrypsin c, metridin, trypsin, thrombin, coagul e, barrierpepsin, bacterial leader peptidase I. lation factor Xa, plasmin, enteropeptidase, acrosin, alpha pseudomonapepsin, plasmepsin. Metallo proteinases include lytic protease, glutamyl, endopeptidase, cathepsin G, atrolysina, microbial collagenase, leucolysin, interstitial col coagulation factor Viia, coagulation factorixa, cucumisi, pro lagenase, neprilysin, envely sin, iga-specific metalloendopep lyl oligopeptidase, coagulation factor Xia, brachyurin, plasma tidase, procollagen N-endopeptidase, thimet oligopeptidase, kallikrein, tissue kallikrein, pancreatic elastase, leukocyte neurolysin, stromelysin 1, meprinA, procollagen C-endopep elastase, coagulation factor Xia, chymase, complement com tidase, peptidyl-lys metalloendopeptidase, astacin, stromel ponent clrS5, complement component clss5, classical ysin, 2., matrilysin gelatinase, aeromonolysin, pseudolysin, complement pathway c3/c5 convertase, complement factor I. thermolysin, bacillolysin, aureolysin, coccolysin, mycolysin, complement factor D, alternative-complement pathway c3/c5 beta-lytic metalloendopeptidase, peptidyl-asp metalloen convertase, cerevisin, hypodermin C, lysyl endopeptidase, 25 dopeptidase, neutrophil collagenase, gelatinase B, leish endopeptidase 1a, gamma-reni, Venombin ab, leucyl manolysin, saccharolysin, autolysin, deuterolysin, serralysin, endopeptidase, tryptase, Scutelarin, kexin, Subtilisin, oryzin, atrolysin B, atrolysin C. atroxase, atrolysin E. atrolysin F. endopeptidase k, thermomycolin, thermitase, endopeptidase adamalysin, horrilysin, ruberlysin, bothropasin, bothrolysin, SO, T-plasminogen activator, protein C, pancreatic endopep ophiolysin, trimerelysin I, trimerelysin II, mucrolysin, pitril tidase E, pancreatic elastase ii, IGA-specific serine endopep 30 ysin, insulysin, O-Syaloglycoprotein endopeptidase, russell tidase, U-plasminogen, activator, Venombin A, furin, myelo ysin, mitochondrial, intermediate, peptidase, dactylysin, nar blastin, semenogelase, granzyme A or cytotoxic dilysin, magnolysin, meprin B, mitochondrial processing T-lymphocyte proteinase 1, granzyme B or cytotoxic T-lym peptidase, macrophage elastase, choriolysin, toxilysin. Pro phocyte proteinase 2, Streptogrisin A, treptogrisin B, teinases of unknown mechanism include thermopsin and glutamylendopeptidase II, oligopeptidase B, limulus clotting multicatalytic endopeptidase complex. TABLE 2 A fluorescens strain MB214 proteases Family ORFID Gene Function Location Aspartic Peptidases A8 (signal peptidase II family) RXFOS383.2 Lipoprotein signal peptidase (ec Cytoplasmic 3.4.23.36) Membrane A24 (type IV prepilin peptidase family) RXFO5379.1 type 4 prepilin peptidase pild (ec Cytoplasmic 3.4.99.—) Membrane Cysteine Peptidases C15 (pyroglutamyl peptidase I family) RXFO2161.1 Pyrrollidone-carboxylate peptidase Cytoplasmic (ec 3.4.19.3) C40 RXFO 1968.1 invasion-associated protein, P60 Signal peptide RXFO4920.1 invasion-associated protein, P60 Cytoplasmic RXFO4923.1 phosphatase-associated protein Signal peptide papq C56 (PfpI endopeptidase family) RXFO1816.1 protease I (ec 3.4.—.—) Non-secretory Metallopeptidases

M1 RXFO8773.1 Membrane alanine aminopeptidase Non-secretory (ec 3.4.11.2) M3 RXFOOS 61.2 prlC Oligopeptidase A (ec 3.4.24.70) Cytoplasmic RXFO4631.2 Zn-dependent oligopeptidases Cytoplasmic M4 (thermolysin family) RXFOS113.2 Extracellular metalloprotease Extracellular precursor (ec 3.4.24.—) M41 (FtsH endopeptidase family) RXFOS400.2 Cell division protein fish (ec Cytoplasmic 3.4.24.—) Membrane M10 RXFO43O4.1 Serralysin (ec 3.4.24.40) Extracellular RXFO4SOO.1 Serralysin (ec 3.4.24.40) Extracellular RXFO1590.2 Serralysin (ec 3.4.24.40) Extracellular RXFO4495.2 Serralysin (ec 3.4.24.40) Extracellular RXFO2796.1 Serralysin (ec 3.4.24.40) Extracellular US 9,394,571 B2 15 16 TABLE 2-continued A fluorescens Strain MB214 proteases Family ORFID Gene Function Location M14 (carboxypeptidase A family) RXFO9091.1 Zinc-carboxypeptidase precursor Cytoplasmic (ec 3.4.17.—) M16 (pitrilysin family) RXFO3441.1 Coenzyme pad synthesis protein F Non-secretory (ec 3.4.99.—) RXFO 1918.1 Zinc protease (ec 3.4.99.—) Signal peptide RXFO 1919.1 Zinc protease (ec 3.4.99.—) Periplasmic RXFO3699.2 processing peptidase (ec 3.4.24.64) Signal peptide M17 (leucyl aminopeptidase family) RXFOO285.2 Cytosol aminopeptidase (ec Non-secretory 3.4.11.1) M18 RXFO7879.1 Aspartyl aminopeptidase (ec Cytoplasmic 3.4.11.21) M2O RXFOO811.1 dapF. Succinyl-diaminopimelate Cytoplasmic desuccinylase (ec 3.5.1.18) RXFO4052.2 Xaa-His dipeptidase (ec 3.4.13.3) Signal peptide RXFO1822.2 Carboxypeptidase G2 precursor (ec Signal peptide 3.4.17.11) RXFO9831.2:: N-acyl-L-amino acid Signal peptide RXFO4892.1 amidohydrolase (ec 3.5.1.14) M28 (aminopeptidase Y family) RXFO3488.2 Alkaline phosphatase isozyme OuterMembrane conversion protein precursor (ec 3.4.11.—) M42 (glutamyl aminopeptidase family) RXFO5615.1 Deblocking aminopeptidase (ec Non-secretory 3.4.11.—) M22 RXFOS817.1 O-Sialoglycoprotein endopeptidase Extracellular (ec 3.4.24.57) RXFO3065.2 Glycoprotease protein family Non-secretory M23 RXFO1291.2 Cell wall endopeptidase, family Signal peptide M23, M37 RXFO3916. Membrane proteins related to Signal peptide metalloendopeptidases RXFO9147.2 Cell wall endopeptidase, family Signal peptide M23, M37 M24 RXFO4693. Methionine aminopeptidase (ec Cytoplasmic 3.4.11.18) RXFO3364. Methionine aminopeptidase (ec Non-secretory 3.4.11.18) RXFO2980. Xaa-Pro aminopeptidase (ec Cytoplasmic 3.4.11.9) RXFO6564. Xaa-Pro aminopeptidase (ec Cytoplasmic 3.4.11.9) M48 (Ste24 endopeptidase family) RXFO5137. Heat shock protein HtpX Cytoplasmic Membrane RXFOSO81. Zinc metalloprotease (ec 3.4.24.—) Signal peptide M50 (S2P protease family) RXFO4692. Membrane metalloprotease Cytoplasmic Membrane Serine Peptidases S1 (chymotrypsin family) RXFO1250.2 protease do (ec 3.4.21.—) Periplasmic RXFO7210.1 protease do (ec 3.4.21.—) Periplasmic S8 (subtilisin family) RXFO6755.2 serine protease (ec 3.4.21.—) Non-secretory RXFO8517.1 serine protease (ec 3.4.21.—) Extracellular RXFO8627.2 extracellular serine protease (ec Signal peptide 3.4.21.—) RXFO6281.1 Extracellular serine protease Non-secretory precursor (ec 3.4.21.—) RXFO8978.1 extracellular serine protease (ec OuterMembrane 3.4.21.—) RXFO6451.1 serine protease (ec 3.4.21.—) Signal peptide S9 (prolyl oligopeptidase family) RXFO2OO3.2 Protease ii (ec 3.4.21.83) Periplasmic RXFOO458.2 Hydrolase Non-secretory S11 (D-Ala-D-Ala carboxypeptidase RXFO4657.2 D-alanyl-D-alanine-endopeptidase Periplasmic A family) (ec 3.4.99.—) RXFOO670.1 D-alanyl-D-alanine Cytoplasmic carboxypeptidase (ec 3.4.16.4) Membrane S13 (D-Ala-D-Ala peptidase C family) RXFOO133.1 D-alanyl-meso-diaminopimelate OuterMembrane endopeptidase (ec 3.4.——) RXFO4960.2 D-alanyl-meso-diaminopimelate Signal peptide endopeptidase (ec 3.4.——) S14 (ClpP endopeptidase family) RXFO4567.1 clpP atp-dependent Clp protease Non-secretory proteolytic subunit (ec 3.4.21.92) RXFO4663.1 clpP atp-dependent Clp protease Cytoplasmic proteolytic subunit (ec 3.4.21.92) S16 (Ion protease family) RXFO4653.2 atp-dependent protease La (ec Cytoplasmic 3.4.21.53) RXFO8653.1 atp-dependent protease La (ec Cytoplasmic 3.4.21.53) US 9,394,571 B2 17 18 TABLE 2-continued A fluorescens strain MB214 proteases Family ORFID Gene Function Location RXFOS943. atp-dependent protease La (ec Cytoplasmic 3.4.21.53) S24 (LexA family) RXFOO449. Lex A repressor (ec 3.4.21.88) Non-secretory RXFO3397. Lex A repressor (ec 3.4.21.88) Cytoplasmic S26 (signal peptidase I family) RXFO1181. Signal peptidase I (ec 3.4.21.89) Cytoplasmic Membrane S33 RXFOS236. pip3 Proline iminopeptidase (ec 3.4.11.5) Non-secretory RXFO48O2. pip1 Proline iminopeptidase (ec 3.4.11.5) Non-secretory RXFO4808.2 pip2 Proline iminopeptidase (ec 3.4.11.5) Cytoplasmic S41 (C-terminal processing RXFO6586. Tail-specific protease (ec 3.4.21.—) Signal peptide peptidase family) RXFO 1037. Tail-specific protease (ec 3.4.21.—) Signal peptide S45 RXFO7170. pacB2 Penicillin acylase (ec 3.5.1.11) Signal peptide RXFO 6399.2 pacB1 Penicillin acylase ii (ec 3.5.1.11) Signal peptide S49 (protease IV family) RXFO6993.2 possible protease Sohlb (ec 3.4.—.—) Non-secretory RXFO1418. protease iv (ec 3.4.——) Non-secretory S58 (Dmp A aminopeptidase family) RXFO63O8.2 D-aminopeptidase (ec 3.4.11.19) Cytoplasmic Membrane Threonine Peptidases T1 (proteasome family) RXFO 1961.2 hslV atp-dependent protease hslV (ec Cytoplasmic 3.4.25.—) T3 (gamma-glutamyltransferase family) RXFO2342.1 ggt1 Gamma-glutamyltranspeptidase (ec Periplasmic 2.3.2.2) RXFO4424.2 ggt2 Gamma-glutamyltranspeptidase (ec Periplasmic 2.3.2.2) Unclassified Peptidases U32 RXFOO428.1 protease (ec 3.4.——) Cytoplasmic RXFO2151.2 protease (ec 3.4.——) Cytoplasmic U61 RXFO4715.1 Muramoyltetrapeptide Non-secretory carboxypeptidase (ec 3.4.17.13) U62 RXFO4971.2 pmbA PmbA protein Cytoplasmic RXFO4968.2 TldD protein Cytoplasmic Non MEROPS Proteases RXFOO325.1 Repressor protein C2 Non-secretory RXFO2689.2 Microsomal dipeptidase (ec Cytoplasmic 3.4.13.19) RXFO2739.1 membrane dipeptidase (3.4.13.19) Signal peptide RXFO3329.2 Hypothetical Cytosolic Protein Cytoplasmic RXFO2492.1 Xaa-Pro dipeptidase (ec 3.4.13.9). Cytoplasmic RXFO4047.2 caax amino terminal protease Cytoplasmic amily Membrane RXFO8136.2 protease (transglutaminase-like Cytoplasmic protein) RXFO9487.1 Zinc metalloprotease (ec 3.4.24.—) Non-secretory

Additional Protein Modification Enzymes TABLE 3 In another embodiment, the target gene comprises a gene involved in proper protein processing and/or modification. 50 Classes of enzymes involved in protein processing Common modifications include disulfide bond formation, glycosylation, acetylation, acylation, phosphorylation, and Class Examples gamma-carboxylation, all of which can regulate protein fold Glycosyltransferases C-glucan-branching glycosyltransferase ing and biological activity. A non-exhaustive list of several (EC 2.4.1.18) enzymatic branching factor classes of enzymes involved in protein processing is found in 55 branching glycosyltransferase enzyme Q Table 3. One of skill in the art will recognize how to identify glucosan transglycosylase a target gene useful in the host cell chosen for the array, or glycogen branching enzyme useful with the heterologous protein of interest, from among amylose isomerase the classes of protein modification enzymes listed in Table 3. plant branching enzyme The target gene may be endogenous to the host cell utilized, 60 C-1,4-glucan:C-1,4-glucan-6- may be endogenous to the organism from which the heterolo glycosyltransferase starch branching enzyme gous protein of interest is derived, or may be known to facili UDP-N-acetyl-D-galactosamine:polypeptide tate proper processing of a heterologously expressed protein N-acetylgalactosaminyltransferase of interest. It is also recognized that any gene involved in 65 GDP-fucose protein O-fucosyltransferase 2 protein production can be targeted according to desired speci O-GlcNAc transferase fications for the heterologous protein of interest. US 9,394,571 B2 19 20 TABLE 3-continued tein or regulatory sequence that modulate transcription of an existing target sequence can also be inserted. Classes of enzymes involved in protein processing Genome Modification Class Examples The genome of the host cell can be modified via a genetic targeting event, which can be by insertion or recombination, Histone acetyltransferase nucleosome-histone acetyltransferase for example homologous recombination. Homologous (EC 2.3.1.48) histone acetokinase histone acetylase recombination refers to the process of DNA recombination histone transacetylase based on sequence homology. Homologous recombination histone deacetylase permits site-specific modifications in endogenous genes and Protein kinase (EC 2.7) non-specific serine/threonine protein kinase 10 Fas-activated serine/threonine kinase thus novel alterations can be engineered into a genome (see, Goodpasture antigen-binding protein kinase for example Radding (1982) Ann. Rev. Genet. 16: 405; U.S. IKB kinase cAMP-dependent protein kinase Pat. No. 4,888,274). cGMP-dependent protein kinase Various constructs can be prepared for homologous recom protein kinase C 15 binationata target locus. Usually, the construct can include at polo kinase cyclin-dependent kinase least 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 70 bp, 100 bp, 500 bp, mitogen-activated protein kinase 1 kbp. 2 kbp. 4 kbp. 5 kbp. 10 kbp. 15 kbp. 20 kbp, or 50 kbp mitogen-activated protein kinase kinase kinase of sequence homologous with the identified locus. Various receptor protein serine/threonine kinase dual-specificity kinase considerations can be involved in determining the extent of Phosphatase protein-tyrosine-phosphatase 2O homology of target gene sequences, such as, for example, the (EC 3.1.3.48) phosphotyrosine phosphatase size of the target locus, availability of sequences, relative phosphoprotein phosphatase (phosphotyrosine) efficiency of double cross-over events at the target locus and phosphotyrosine histone phosphatase protein phosphotyrosine phosphatase the similarity of the target sequence with other sequences. tyrosylprotein phosphatase The modified gene can include a sequence in which DNA phosphotyrosine protein phosphatase 25 Substantially isogenic flanks the desired sequence modifica phosphotyrosylprotein phosphatase tyrosine O-phosphate phosphatase tions with a corresponding target sequence in the genome to PPT-phosphatase be modified. The “modified gene' is the sequence being intro PTPase duced into the genome to alter the expression of a protease or phosphotyrosine protein phosphatase a protein folding modulator in the host cell. The “target gene’ PTP-phosphatase 30 is the sequence that is being replaced by the modified gene. The substantially isogenic sequence can be at least about Methods for Modulating the Expression of Target Genes 95%,97-98%, 99.0-99.5%, 99.6-99.9%, or 100% identical to One or more host cell populations of the array can be the corresponding target sequence (except for the desired modified by any technique known in the art, for example by a sequence modifications). The modified gene and the targeted technique wherein at least one target gene is knocked out of gene can share stretches of DNA at least about 10, 20, 30, 50. the genome, or by mutating at least one target gene to reduce 75, 150 or 500 base pairs that are 100% identical. expression of the gene, by altering at least one promoter of at Nucleotide constructs can be designed to modify the least one target gene to reduce expression of the target gene, endogenous, target gene product. The modified gene or by coexpressing (with the heterologous protein or polypep 40 sequence can have one or more deletions, insertions, Substi tide of interest) the target gene or an inhibitor of the target tutions or combinations thereof designed to disrupt the func gene in the host genome. As discussed Supra, the target gene tion of the resultant gene product. In one embodiment, the can be endogenous to the host cell populations in the array, or alteration can be the insertion of a selectable marker gene can be heterologously expressed in each of the host cell fused in reading frame with the upstream sequence of the populations. 45 target gene. The expression of target genes can be increased, for The genome can also be modified using insertional inacti example, by introducing into at least one cell in a host popu Vation. In this embodiment, the genome is modified by lation an expression vector comprising one or more target recombining a sequence in the gene that inhibits gene product genes involved in protein production. The target gene expres formation. This insertion can either disrupt the gene by insert sion can also be increased, for example, by mutating a pro 50 ing a separate element, or remove an essential portion of the moter of a target gene. A host cell or organism that expresses gene. In one embodiment, the insertional deletion also a heterologous protein can also be genetically modified to includes insertion of a gene coding for resistance to a particu increase the expression of at least one target gene involved in lar stressor, such as an antibiotic, or for growth in a particular protein production and decrease the expression of at least one media, for example for production of an essential amino acid. 55 The genome can also be modified by use of transposons, target gene involved in protein degradation. which are genetic elements capable of inserting at sites in The genome may be modified to modulate the expression prokaryote genomes by mechanisms independent of homolo of one or more target genes by including an exogenous gene gous recombination. Transposons can include, for example, or promoter element in the genome or in the host with an TnT, Tn5, or Tn 10 in E. coli, Tn554 in S. aureus, IS900 in M. expression vector, by enhancing the capacity of a particular 60 paratuberculosis, IS492 from Pseudomonas atlantica, IS116 target gene to produce mRNA or protein, by deleting or from Streptomyces and IS900 from M. paratuberculosis. disrupting a target gene or promoter element, or by reducing Steps believed to be involved in transposition include cleav the capacity of a target gene to produce mRNA or protein. The age of the end of the transposonto yield 3'OH; strand transfer, genetic code can be altered, thereby affecting transcription in which transposase brings together the 3'OH exposed end of and/or translation of a target gene, for example through Sub- 65 transposon and the identified sequence; and a single step stitution, deletion ("knock-out'), co-expression, or insertion transesterification reaction to yield a covalent linkage of the ("knock-in”) techniques. Additional genes for a desired pro transposon to the identified DNA. The key reaction per US 9,394,571 B2 21 22 formed by transposase is generally thought to be nicking or perature sensitive for DNA replication. After transformation Strand exchange, the rest of the process is done by host of the plasmid into the appropriate host, it is possible to select enzymes. for integration of the plasmid into the chromosome at 44°C. In one embodiment, the expression or activity of a target Subsequent growth of these cointegrates at 30° C. leads to a gene or protein is increased by incorporating a genetic second recombination event, resulting in their resolution. sequence encoding the target protein or homolog thereof into Depending on where the second recombination event takes the genome by recombination. In another embodiment, a place, the chromosome will either have undergone a gene promoter is inserted into the genome to enhance the expres replacement or retain the original copy of the gene. sion of the target gene or homolog. In another embodiment, Other strategies have been developed to inhibit expression the expression or activity of a target gene or homolog thereof 10 of particular gene products. For example, RNA interference is decreased by recombination with an inactive gene. In (RNAi), particularly using small interfering RNA (siRNA), another embodiment, a sequence that encodes a different has been extensively developed to reduce or even eliminate gene, which can have a separate function in the cell or can be expression of a particular gene product. siRNAS are short, a reporter gene Such as a resistance marker or an otherwise double-stranded RNA molecules that can target complemen detectable marker gene, can be inserted into the genome 15 tary mRNAs for degradation. RNAi is the phenomenon in through recombination. In yet another embodiment, a copy of which introduction of a double-stranded RNA suppresses the at least a portion of the target gene that has been mutated at expression of the homologous gene. dsRNA molecules are one or more locations is inserted into the genome through reduced in vivo to 21-23 nt siRNAs which are the mediators recombination. The mutated version of the target gene may of the RNAi effect. Upon introduction, double stranded not encode a protein, or the protein encoded by the mutated RNAs get processed into 20-25 nucleotide siRNAs by an gene may be rendered inactive, the activity may be modulated RNase III-like enzyme called Dicer (initiation step). Then, (either increased or decreased), or the mutant protein can have the siRNAS assemble into endoribonuclease-containing com a different activity when compared to the native protein. plexes known as RNA-induced silencing complexes (RISCs), There are strategies to knock out genes in bacteria, which unwinding in the process. The siRNA strands Subsequently have been generally exemplified in E. coli. One route is to 25 guide the RISCs to complementary RNA molecules, where clone a gene-internal DNA fragment into a vector containing they cleave and destroy the cognate RNA (effecter step). an antibiotic resistance gene (e.g. amplicillin). Before cells are Cleavage of cognate RNA takes place near the middle of the transformed via conjugative transfer, chemical transforma region bound by the siRNA strand. RNAi has been success tion or electroporation (Puehler, et al. (1984) Advanced fully used to reduce gene expression in a variety of organisms Molecular Genetics New York, Heidelberg, Berlin, Tokyo, 30 including Zebrafish, nematodes (C. elegans), insects (Droso Springer Verlag), an origin of replication, Such as the vegeta phila melanogaster), planaria, cnidaria, trypanosomes, mice tive plasmid replication (the oriV locus) is excised and the and mammalian cells. remaining DNA fragment is re-ligated and purified (Sam The genome can also be modified by mutation of one or brook, et al. (2000) Molecular cloning: A laboratory manual, more nucleotides in an open reading frame encoding a target third edition Cold Spring Harbor, N.Y., Cold Spring Harbor 35 gene. Techniques for genetic mutation, for instance site Laboratory Press). Alternatively, antibiotic-resistant plas directed mutagenesis, are well known in the art. Some mids that have a DNA replication origin can be used. After approaches focus on the generation of random mutations in transformation, the cells are plated onto e.g. LB agar plates chromosomal DNA such as those induced by X-rays and containing the appropriate antibiotics (e.g. 200 micrograms/ chemicals. mL amplicillin). Colonies that grow on the plates containing 40 Coexpression the antibiotics presumably have undergone a single recombi In one embodiment, one or more target genes in the host nation event (Snyder, L., W. Champness, et al. (1997) cell can be modified by including one or more vectors that Molecular Genetics of Bacteria Washington D.C., ASM encode the target gene(s) to facilitate coexpression of the Press) that leads to the integration of the entire DNA fragment target gene with the heterologous protein or peptide. In into the genome at the homologous locus. Further analysis of 45 another embodiment, the host cell is modified by enhancing a the antibiotic-resistant cells to verify that the desired gene promoter for a target gene, including by adding an exogenous knock-out has occurred at the desired locus is e.g. by diag promoter to the host cell genome. nostic PCR (McPherson, M.J., P. Quirke, et al. (1991) PCR: In another embodiment, one or more target genes in the A Practical Approach New York, Oxford University Press). host cell is modified by including one or more vectors that Here, at least two PCR primers are designed: one that hybrid 50 encode an inhibitor of a target gene. Such as a protease inhibi izes outside the DNA region that was used for the construc torto inhibit the activity of a target protease. Such an inhibitor tion of the gene knock-out; and one that hybridizes within the can be an antisense molecule that limits the expression of the remaining plasmid backbone. Successful PCR amplification target gene, a cofactor of the target gene or a homolog of the of the DNA fragment with the correct size followed by DNA target gene. Antisense is generally used to refer to a nucleic sequence analysis will verify that the gene knock-out has 55 acid molecule with a sequence complementary to at least a occurred at the correct location in the bacterial chromosome. portion of the target gene. In addition, the inhibitor can be an The phenotype of the newly constructed mutant Strain can interfering RNA or a gene that encodes an interfering RNA. In then be analyzed by, e.g., SDS polyacrylamide gel electro Eukaryotic organisms, such an interfering RNA can be a phoresis (Simpson, R.J. (2003) Proteins and Proteomics—A small interfering RNA or a ribozyme, as described, for Laboratory Manual. Cold Spring Harbor, N.Y., Cold Spring 60 example, in Fire, A. etal. (1998) Nature 391:806-11, Elbashir Harbor Laboratory Press). et al. (2001) Genes & Development 15(2):188-200, Elbashir An alternate route to generate a gene knock-out is by use of et al. (2001) Nature 411 (6836):494-8, U.S. Pat. No. 6,506, a temperature-sensitive replicon, such as the pSC101 replicon 559 to Carnegie Institute, U.S. Pat. No. 6,573,099 to Benitec, to facilitate gene replacement (Hamilton, et al. (1989) Jour U.S. patent application Nos. 2003/0108923 to the Whitehead nal of Bacteriology 171 (9): 4617-22). The process proceeds 65 Inst., and 2003/0114409, PCT Publication Nos. WOO3/ by homologous recombination between a gene on a chromo 006477, WO03/012052, WO03/023015, WO03/056022, Some and homologous sequences carried on a plasmid tem WOO3/O64621 and WOO3/07O966. US 9,394,571 B2 23 24 The inhibitor can also be another protein or peptide. The achieved. Various modifications can be made to the sequence, inhibitor can, for example, be a peptide with a consensus to allow for restriction analysis, excision, identification of sequence for the target protein. The inhibitor can also be a probes, etc. Silent mutations can be introduced, as desired. At protein or peptide that can produce a director indirect inhibi various stages, restriction analysis, sequencing, amplification tory molecule for the target protein in the host. For example, with the polymerase chain reaction, primer repair, in vitro protease inhibitors can include Amastatin, E-64, Antipain, mutagenesis, etc. can be employed. Processes for the incor Elastatinal, APMSF, Leupeptin, Bestatin, Pepstatin, Benza poration of antibiotic resistance genes and negative selection midine, 1,10-Phenanthroline, Chymostatin, Phosphorami factors will be familiar to those of ordinary skill in the art (see, don, 3,4-dichloroisocoumarin, TLCK, DFPTPCK. Over 100 e.g., WO 99/15650; U.S. Pat. No. 6,080,576; U.S. Pat. No. naturally occurring protein protease inhibitors have been 10 identified so far. They have been isolated in a variety of 6,136,566; Niwa, et al., J. Biochem. 113:343-349 (1993); and organisms from bacteria to animals and plants. They behave Yoshida, et al., Transgenic Research, 4:277-287 (1995)). as tight-binding reversible or pseudo-irreversible inhibitors The construct can be prepared using a bacterial vector, of proteases preventing Substrate access to the active site including a prokaryotic replication system, e.g. an origin through steric hindrance. Their size are also extremely vari 15 recognizable by a prokaryotic cell Such as P. fluorescens or E. able from 50 residues (e.g. BPTI: Bovine Pancreatic Trypsin coli. A marker, the same as or different from the marker to be Inhibitor) to up to 400 residues (e.g. alpha-1PI: alpha-1 Pro used for insertion, can be employed, which can be removed teinase Inhibitor). They are strictly class-specific except pro prior to introduction into the host cell. Once the vector con teins of the alpha-macroglobulin family (e.g. alpha-2 macro taining the construct has been completed, it can be further globulin) which bind and inhibit most proteases through a manipulated, such as by deletion of certain sequences, linear molecular trap mechanism. ization, or by introducing mutations, deletions or other An exogenous vector or DNA construct can be transfected sequences into the homologous sequence. In one embodi or transformed into the host cell. Techniques for transfecting ment, the target gene construct and the heterologous protein and transforming eukaryotic and prokaryotic cells respec construct are part of the same expression vector, and may or tively with exogenous nucleic acids are well known in the art. 25 may not be under the control of the same promoter element. In These can include lipid vesicle mediated uptake, calcium another embodiment, they are on separate expression vectors. phosphate mediated transfection (calcium phosphate/DNA After final manipulation, the construct can be introduced into co-precipitation), viral infection, particularly using modified the cell. viruses such as, for example, modified adenoviruses, micro Cell Growth Conditions injection and electroporation. For prokaryotic transforma 30 The cell growth conditions for the host cells described tion, techniques can include heat shock mediated uptake, herein include that which facilitates expression of the protein bacterial protoplast fusion with intact cells, microinjection of interest in at least one strain in the array (or at least a and electroporation. Techniques for plant transformation proportion of cells thereof), and/or that which facilitates fer include Agrobacterium mediated transfer, such as by A. tume mentation of the expressed protein of interest. As used herein, faciens, rapidly propelled tungsten or gold microprojectiles, 35 the term "fermentation' includes both embodiments in which electroporation, microinjection and polyethylene glycol literal fermentation is employed and embodiments in which mediated uptake. The DNA can be single or double stranded, other, non-fermentative culture modes are employed. linear or circular, relaxed or supercoiled DNA. For various Growth, maintenance, and/or fermentation of the populations techniques for transfecting mammalian cells, see, for of host cells in the array may be performed at any scale. example, Keown et al. (1990) Processes in Enzymology Vol. 40 However, where multiple populations of host cells are 185, pp. 527-537. screened simultaneously, the scale will be limited by the An expression construct encoding a target gene or an number of different populations and the capacity to grow and enhancer or inhibitor thereof can be constructed as described test multiple populations of host cells. In one embodiment, below for the expression constructs comprising the heterolo the fermentation medium may be selected from among rich gous protein or polypeptide of interest. For example, the 45 media, minimal media, and mineral salts media. In another constructs can contain one, or more than one, internal ribo embodiment either a minimal medium or a mineral salts some entry site (IRES). The construct can also contain a medium is selected. In still another embodiment, a minimal promoter operably linked to the nucleic acid sequence encod medium is selected. In yet another embodiment, a mineral ing at least a portion of the target gene, or a cofactor of the salts medium is selected. target gene, a mutant version of at least a portion of the target 50 Mineral salts media consists of mineral salts and a carbon gene, or in some embodiments, an inhibitor of the target gene. Source Such as, e.g., glucose, Sucrose, or glycerol. Examples Alternatively, the construct can be promoterless. In cases in of mineral salts media include, e.g., M9 medium, Pseudomo which the construct is not designed to incorporate into the nas medium (ATCC 179), Davis and Mingioli medium (see, cellular DNA/genome, the vector typically contains at least BD Davis & E S Mingioli (1950) in J. Bact. 60:17-28). The one promoter element. In addition to the nucleic acid 55 mineral salts used to make mineral salts media include those sequences, the expression vector can contain selectable selected from among, e.g., potassium phosphates, ammo marker sequences. The expression constructs can further con nium Sulfate or chloride, magnesium Sulfate or chloride, and tain sites for transcription initiation, termination, and/or ribo trace minerals such as calcium chloride, borate, and Sulfates Some binding sites. The identified constructs can be inserted of iron, copper, manganese, and zinc. No organic nitrogen into and can be expressed in any prokaryotic or eukaryotic 60 Source. Such as peptone, tryptone, amino acids, or a yeast cell, including, but not limited to bacterial cells, such as P. extract, is included in a mineral salts medium. Instead, an fluorescens or E. coli, yeast cells, mammalian cells, such as inorganic nitrogen source is used and this may be selected CHO cells, or plant cells. from among, e.g., ammonium salts, aqueous ammonia, and The construct can be prepared in accordance with pro gaseous ammonia. A preferred mineral salts medium will cesses known in the art. Various fragments can be assembled, 65 contain glucose as the carbon Source. In comparison to min introduced into appropriate vectors, cloned, analyzed and eral salts media, minimal media can also contain mineral salts then manipulated further until the desired construct has been and a carbon Source, but can be supplemented with, e.g., low US 9,394,571 B2 25 26 levels of amino acids, vitamins, peptones, or other ingredi humidity of the incubation is controlled to minimize evapo ents, though these are added at very minimal levels. ration from the culture vessel, and permit the use of smaller In one embodiment, media can be prepared using the com Volumes. Alternatively, or in addition to controlling humidity, ponents listed in Table 4 below. The components can be added the vessels may be covered with lids in order to minimize in the following order: first (NH)HPO, KHPO and citric evaporation. Selection of the incubation temperature depends acid can be dissolved in approximately 30 liters of distilled primarily upon the identity of the host cells utilized. Selection water; then a solution of trace elements can be added, fol of the percent humidity to control evaporation is based upon lowed by the addition of an antifoam agent, such as Ucolub N the selected volume of the vessel and concentration and Vol 115. Then, after heat sterilization (such as at approximately ume of the cell culture in the vessel, as well as upon the 10 incubation temperature. Thus, the humidity may vary from 121° C.), sterile solutions of glucose MgSO and thiamine about 10% to about 80%. It should be understood that selec HCL can be added. Control of pH at approximately 6.8 can be tion of a suitable conditions is well within the skill of the art. achieved using aqueous ammonia. Sterile distilled water can Screening then be added to adjust the initial volume to 371 minus the The strain array described herein can be screened for the glycerol stock (123 mL). The chemicals are commercially 15 optimal host cell population in which to express a heterolo available from various Suppliers, such as Merck. gous protein of interest. The optimal host cell population can be identified or selected based on the quantity, quality, and/or TABLE 4 location of the expressed protein of interest. In one embodi Medium composition ment, the optimal host cell population is one that results in an increased yield of the protein or polypeptide of interest within Initial concentration the host cell compared to other populations of phenotypically Component distinct host cells in the array. The increased production alternatively can be an increased KH2PO 13.3 g level of properly processed protein or polypeptide per gram of (NH4)2HPO 4.0 g 25 protein produced, or per gram of host protein. The increased Citric Acid 1.7 g l' MgSO 7H2O 1.2 g I' production can also be an increased level of recoverable pro Trace metal solution 10 ml || tein or polypeptide produced per gram of heterologous pro Thiamin HCI 4.5 mg l' tein or per gram of host cell protein. The increased production Glucose-H2O 27.3 g || Antifoam Ucolub N115 0.1 ml | can also be any combination of an increased level of total 30 protein, increased level of properly processed or properly Feeding solution folded protein, or increased level of active or soluble protein. MgSO 7H2O 19.7 g || In this embodiment, the term “increased” or “improved” is Glucose-H2O 770 g || relative to the level of protein or polypeptide that is produced, NH 23 g Trace metal solution properly processed, soluble, and/or recoverable when the 35 protein or polypeptide of interest is expressed in one or more 6 g | Fe(III) citrate 1.5 g I' MnCl2 4H2O other populations of host cells in the array. The increased 0.8g | ZmCHCOOI 2H2O 0.3 g production may optimize the efficiency of the cell or organ HBO 0.25 g I Na2MoC—2H2O 0.25 g l'CoCl2 ism by for example, decreasing the energy expenditure, 6H2O increasing the use of available resources, or decreasing the 0.15g | CuCl2.H2O 0.84 g lethylene 40 requirements for growth supplements in growth media. The Dinitrilo-tetracetic acid Nasah 2H2O increased production may also be the result of a decrease in (Tritriplex III, Merck) proteolyic degradation of the expressed protein. In one embodiment, at least one strain in the array produces In the present invention, growth, culturing, and/or fermen at least 0.1 mg/ml correctly processed protein. A correctly tation of the transformed host cells is performed within a 45 processed protein has anamino terminus of the native protein. temperature range permitting Survival of the host cells, pref In another embodiment, at least one strain produces 0.1 to 10 erably a temperature within the range of about 4°C. to about mg/ml correctly processed protein in the cell, including at 55°C., inclusive. Thus, e.g., the terms “growth' (and “grow.” least about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, “growing”), “culturing (and “culture'), and “fermentation' about 0.7, about 0.8, about 0.9 or at least about 1.0 mg/ml (and “ferment,” “fermenting'), as used herein in regard to the 50 correctly processed protein. In another embodiment, the total host cells of the present invention, inherently means correctly processed protein or polypeptide of interest pro “growth.” “culturing.” and “fermentation.” within a tempera duced by at least one strain in the array is at least 1.0 mg/ml. ture range of about 4°C. to about 55° C., inclusive. In addi at least about 2 mg/ml, at least about 3 mg/ml, about 4 mg/ml. tion, “growth' is used to indicate both biological states of about 5 mg/ml, about 6 mg/ml, about 7 mg/ml, about 8 mg/ml. active cell division and/or enlargement, as well as biological 55 about 10 mg/ml, about 15 mg/ml, about 20 mg/ml, about 25 states in which a non-dividing and/or non-enlarging cell is mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml. being metabolically sustained, the latter use of the term about 45 mg/ml, at least about 50 mg/ml, or greater. In some “growth' being synonymous with the term “maintenance.” embodiments, the amount of correctly processed protein pro The host cells of the array should be grown and maintained duced is at least about 5%, about 10%, about 15%, about 20%, at a suitable temperature for normal growth of that cell type. 60 about 25%, about 30%, about 40%, about 50%, about 60%, Such normal growth temperatures may be readily selected about 70%, about 80%, about 90%, about 95%, about 96%, based on the known growth requirements of the selected host about 97%, about 98%, at least about 99%, or more of total cell. Preferably, during the establishment of the culture and heterologous protein in a correctly processed form. particularly during course of the screening, the cell culture is An improved expression of a protein or polypeptide of incubated in a controlled CO/N humidity suitable for 65 interest can also refer to an increase in the solubility of the growth of the selected cells before and after transformation protein. The protein or polypeptide of interest can be pro with the heterologous protein or polypeptide of interest. The duced and recovered from the cytoplasm, periplasm or extra US 9,394,571 B2 27 28 cellular medium of the host cell. The protein or polypeptide that permeabilize the outer cell membrane, including: colicin can be insoluble or soluble. The protein or polypeptide can (Mikschet al. (1997) Arch. Microbiol. 167: 143-150); growth include one or more targeting (e.g., signal or leader) rate (Shokrietal. (2002) App Miocrobiol Biotechnol 58:386 sequences or sequences to assist purification, as discussed 392); Toll II overexpression (Wan and Baneyx (1998) Protein Supra. 5 Expression Purif 14: 13-22); bacteriocin release protein The term “soluble' as used herein means that the protein is (Hsiung et al. (1989) Bio/Technology 7: 267-71), colicin A not precipitated by centrifugation at between approximately lysis protein (Lloubes et al. (1993) Biochimie 75: 451-8) 5,000 and 20,000x gravity when spun for 10-30 minutes in a mutants that leak periplasmic proteins (Furlong and Sund buffer under physiological conditions. Soluble proteins are strom (1989) Developments in Indus. Microbio. 30: 141-8); not part of an inclusion body or other precipitated mass. 10 fusion partners (Jeong and Lee (2002) Appl. Environ. Micro Similarly, “insoluble” means that the protein or polypeptide bio. 68: 4979-4985); or, recovery by osmotic shock (Taguchi can be precipitated by centrifugation at between 5,000 and et al. (1990) Biochimica Biophysica Acta 1049: 278-85). 20,000x gravity when spun for 10-30 minutes in a buffer Transport of engineered proteins to the periplasmic space under physiological conditions. Insoluble proteins or with subsequent localization in the broth has been used to polypeptides can be part of an inclusion body or other pre 15 produce properly folded and active proteins in E. coli (Wan cipitated mass. The term “inclusion body' is meant to include and Baneyx (1998) Protein Expression Purif. 14: 13-22; Sim any intracellular body contained within a cell wherein an mons et al. (2002).J. Immun. Meth. 263: 133-147; Lundell et aggregate of proteins or polypeptides has been sequestered. al. (1990).J. Indust. Microbio. 5: 215-27). In another embodiment, the optimal host cell population The method may also include the step of purifying the produces an increased amount of the protein of interest that is protein or polypeptide of interest from the periplasm or from transported to the periplasm or secreted into the extracellular extracellular media. The heterologous protein or polypeptide space of the host cell. In one embodiment, at least one strain can be expressed in a manner in which it is linked to a tag in the array produces at least 0.1 mg/ml protein in the peri protein and the “tagged” protein can be purified from the cell plasmic compartment. In another embodiment, at least one or extracellular media. strain produces 0.1 to 10 mg/ml periplasmic protein in the 25 In some embodiments, the protein or polypeptide of inter cell, or at least about 0.2, about 0.3, about 0.4, about 0.5, about est can also be produced by at least one strain in the array in 0.6, about 0.7, about 0.8, about 0.9 or at least about 1.0 mg/ml an active form. The term “active” means the presence of periplasmic protein. In one embodiment, the total protein or biological activity, wherein the biological activity is compa polypeptide of interest produced by at least one strain in the rable or substantially corresponds to the biological activity of array is at least 1.0 mg/ml, at least about 2 mg/ml, at least 30 a corresponding native protein or polypeptide. In the context about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml. of proteins this typically means that a polynucleotide or about 7 mg/ml, about 8 mg/ml, about 10 mg/ml, about 15 polypeptide comprises a biological function or effect that has mg/ml, about 20 mg/ml, at least about 25 mg/ml, or greater. In at least about 20%, about 50%, preferably at least about Some embodiments, the amount of periplasmic protein pro 60-80%, and most preferably at least about 90-95% activity duced is at least about 5%, about 10%, about 15%, about 20%, 35 compared to the corresponding native protein or polypeptide about 25%, about 30%, about 40%, about 50%, about 60%, using standard parameters. However, in some embodiments, about 70%, about 80%, about 90%, about 95%, about 96%, it may be desirable to produce a polypeptide that has altered about 97%, about 98%, about 99%, or more of total protein or or improved activity compared to the native protein (e.g., one polypeptide of interest produced. that has altered or improved immunoreactivity, Substrate At least one strain in the array of the invention can also lead 40 specificity, etc). An altered or improved polypeptide may to increased yield of the protein or polypeptide of interest. In result from a particular conformation created by one or more one embodiment, at least one strain produces a protein or of the host cell populations of the array. polypeptide of interest as at least about 5%, at least about The determination of protein or polypeptide activity can be 10%, about 15%, about 20%, about 25%, about 30%, about performed utilizing corresponding standard, targeted com 40%, about 45%, about 50%, about 55%, about 60%, about 45 parative biological assays for particular proteins or polypep 65%, about 70%, about 75%, or greater of total cell protein tides which can be used to assess biological activity. (tcp). “Percent total cell protein' is the amount of protein or The recovery of active protein or polypeptide of interest polypeptide in the host cell as a percentage of aggregate may also be improved in the optimal host strain compared to cellular protein. Methods for the determination of the percent one or more other strains in the array of the invention. Active total cell protein are well known in the art. 50 proteins can have a specific activity of at least about 20%, at In a particular embodiment, at least one host cell popula least about 30%, at least about 40%, about 50%, about 60%, tion in the array can have a heterologous protein production at least about 70%, about 80%, about 90%, or at least about level of at least 1% top and a cell density of at least 40 mg/ml, 95% that of the native protein or polypeptide from which the when grown (i.e. within a temperature range of about 4°C. to sequence is derived. Further, the substrate specificity (k/ about 55° C., including about 10°C., about 15° C., about 20° 55 K) is optionally Substantially similar to the native protein or C., about 25°C., about 30° C., about 35° C., about 40° C., polypeptide. Typically, k/K will be at least about 30%, about 45°C., and about 50°C.) in a mineral salts medium. In about 40%, about 50%, about 60%, about 70%, about 80%, at a particularly preferred embodiment, the expression system least about 90%, at least about 95%, or greater. Methods of will have a protein or polypeptide expression level of at least assaying and quantifying measures of protein and polypep 5% top and a cell density of at least 40 g/L, when grown (i.e. 60 tide activity and Substrate specificity (k/K), are well within a temperature range of about 4°C. to about 55° C. known to those of skill in the art. inclusive) in a mineral salts medium. Measurement of Protein Activity In practice, heterologous proteins targeted to the periplasm The activity of the heterologously-expressed protein or are often found in the broth (see European Patent No. EPO polypeptide of interest can be compared with a previously 288 451), possibly because of damage to or an increase in the 65 established native protein or polypeptide standard activity. fluidity of the outer cell membrane. The rate of this “passive' Alternatively, the activity of the protein or polypeptide of secretion may be increased by using a variety of mechanisms interest can be determined in a simultaneous, or Substantially US 9,394,571 B2 29 30 simultaneous, comparative assay with the native protein or able change in the host cell population. The reporter molecule polypeptide. For example, in vitro assays can be used to can be firefly luciferase and GFP or any other fluorescence determine any detectable interaction between a protein or molecule, as well as beta-galactosidase gene (beta.gal) and polypeptide of interestanda target, e.g. between an expressed chloramphenicol and acetyltransferase gene (CAT). Assays enzyme and Substrate, between expressed hormone and hor for expression produced in conjunction with each of these mone receptor, between expressed antibody and antigen, etc. reporter gene elements are well-known to those skilled in the Such detection can include the measurement of calorimetric art. changes, proliferation changes, cell death, cell repelling, The reporter gene can encode a detectable protein or an changes in radioactivity, changes in solubility, changes in indirectly detectable protein, or the reporter gene can be a molecular weight as measured by gel electrophoresis and/or 10 Survival gene. In a preferred embodiment, the reporterprotein gel exclusion methods, phosphorylation abilities, antibody is a detectable protein. A “detectable protein' or “detection specificity assays such as ELISA assays, etc. In addition, in protein' (encoded by a detectable or detection gene) is a Vivo assays include, but are not limited to, assays to detect protein that can be used as a direct label; that is, the protein is physiological effects of the heterologously expressed protein detectable (and preferably, a cell comprising the detectable or polypeptide in comparison to physiological effects of the 15 protein is detectable) without further manipulation. Thus, in native protein or polypeptide, e.g. weight gain, change in this embodiment, the protein product of the reporter gene electrolyte balance, change in blood clotting time, changes in itself can serve to distinguish cells that are expressing the clot dissolution and the induction of antigenic response. Gen detectable gene. In this embodiment, suitable detectable erally, any in vitro or in vivo assay can be used to determine genes include those encoding autofluorescent proteins. the active nature of the protein or polypeptide of interest that As is known in the art, there are a variety of autofluorescent allows for a comparative analysis to the native protein or proteins known; these generally are based on the green fluo polypeptide so long as Such activity is assayable. Alterna rescent protein (GFP) from Aequorea and variants thereof, tively, the proteins or polypeptides produced in at least one including, but not limited to, GFP. (Chalfie, et al. (1994) strain in the array of the present invention can be assayed for Science 263(5148):802-805); enhanced GFP (EGFP; Clon the ability to stimulate or inhibit interaction between the 25 tech Genbank Accession Number U55762)), blue fluores protein or polypeptide and a molecule that normally interacts cent protein (BFP. Quantum Biotechnologies, Inc., Montreal, with the protein or polypeptide, e.g. a substrate or a compo Canada); Stauber (1998) Biotechniques 24(3):462-471; nent of a signal pathway with which the native protein nor Heim and Tsien (1996) Curr. Biol. 6:178-182), enhanced mally interacts. Such assays can typically include the steps of yellow fluorescent protein (EYFP; Clontech Laboratories, combining the protein with a Substrate molecule under con 30 Inc., Palo Alto, Calif.) and red fluorescent protein. In addition, ditions that allow the protein or polypeptide to interact with there are recent reports of autofluorescent proteins from the target molecule, and detect the biochemical consequence Renilla and Ptilosarcus species. See WO92/15673; WO of the interaction with the protein and the target molecule. 95/07463; WO98/14605; WO 98/26277; WO99/49019; U.S. Assays that can be utilized to determine protein or Pat. No. 5,292,658; U.S. Pat. No. 5,418, 155; U.S. Pat. No. polypeptide activity are described, for example, in Ralph, P. 35 5,683,888; U.S. Pat. No. 5,741,668; U.S. Pat. No. 5,777,079; J., et al. (1984).J. Immunol. 132:1858 or Saiki et al. (1981).J. U.S. Pat. No. 5,804,387: U.S. Pat. No. 5,874,304; U.S. Pat. Immunol. 127:1044, Steward, W. E. II (1980) The Interferon No. 5,876,995; and U.S. Pat. No. 5,925,558; all of which are Systems. Springer-Verlag, Vienna and New York, Broxmeyer, expressly incorporated herein by reference. H. E., et al. (1982) Blood 60:595, Molecular Cloning. A Isolation of Protein or Polypeptide of Interest Laboratory Manua', 2d ed., Cold Spring Harbor Laboratory 40 To measure the yield, Solubility, conformation, and/or Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, activity of the protein of interest, it may be desirable to isolate and Methods in Enzymology. Guide to Molecular Cloning the protein from one or more strains in the array. The isolation Techniques, Academic Press, Berger, S. L. and A. R. Kimmel may be a crude, semi-crude, or pure isolation, depending on eds., 1987, AK Patra et al., Protein Expr Purif, 18(2): p.182 the requirements of the assay used to make the appropriate 92 (2000), Kodama et al., J. Biochem.99: 1465-1472 (1986); 45 measurements. The protein may be produced in the cyto Stewart et al., Proc. Natl. Acad. Sci. USA 90: 5209-5213 plasm, targeted to the periplasm, or may be secreted into the (1993); (Lombillo et al., J. Cell Biol. 128:107-115 (1995); culture or fermentation media. To release proteins targeted to (Vale et al., Cell 42:39-50 (1985). Activity can be compared the periplasm, treatments involving chemicals such as chlo between samples of heterologously expressed protein derived roform (Ames et al. (1984) J. Bacteriol., 160: 1181-1183), from one or more of the other host cell populations in the 50 guanidine-HCl, and Triton X-100 (Naglak and Wang (1990) array, or can be compared to the activity of a native protein, or Enzyme Microb. Technol., 12: 603–611) have been used. both. Activity measurements can be performed on isolated However, these chemicals are not inert and may have detri protein, or can be performed in vitro in the host cell. mental effects on many heterologous protein products or Sub In another embodiment, protein production and/or activity sequent purification procedures. Glycine treatment of E. coli may be monitored directly in the culture by fluorescence or 55 cells, causing permeabilization of the outer membrane, has spectroscopic measurements on, for example, a conventional also been reported to release the periplasmic contents (Ariga microscope, luminometer, or plate reader. Where the protein et al. (1989).J. Ferm. Bioeng. 68: 243-246). The most widely of interest is an enzyme whose Substrate is known, the Sub used methods of periplasmic release of heterologous protein strate can be added to the culture media wherein a fluorescent are osmotic shock (Nosal and Heppel (1966) J. Biol. Chem., signal is emitted when the substrate is converted by the 60 241: 3055-3062; Neu and Heppel (1965) J. Biol. Chem., enzyme into a product. In one embodiment, the expression 240:3685-3692), hen eggwhite (HEW)-lysozyme/ethylene construct encoding the heterologous protein or polypeptide of diamine tetraacetic acid (EDTA) treatment (Neu and Heppel interest further encodes a reported protein. By “reporter pro (1964).J. Biol. Chem., 239:3893-3900: Witholt et al. (1976) tein' is meant a protein that by its presence in or on a cell or Biochim. Biophys. Acta, 443: 534-544: Pierce et al. (1995) when secreted in the media allows the cell to be distinguished 65 ICheme Research. Event, 2:995-997), and combined HEW from a cell that does not contain the reporter protein. Produc lysozyme?osmotic shock treatment (French et al. (1996) tion of the heterologous protein of interest results in a detect Enzyme and Microb Tech., 19:332-338). The French method US 9,394,571 B2 31 32 involves resuspension of the cells in a fractionation buffer trolled expression of lysis genes encoded in T4 phage for the followed by recovery of the periplasmic fraction, where gentle disruption of E. coli cells. osmotic shock immediately follows lysozyme treatment. Upon cell lysis, genomic DNA leaks out of the cytoplasm Typically, these procedures include an initial disruption in into the medium and results in significant increase in fluid osmotically-stabilizing medium followed by selective release 5 Viscosity that can impede the sedimentation of Solids in a in non-stabilizing medium. The composition of these media centrifugal field. In the absence of shear forces such as those (pH, protective agent) and the disruption methods used (chlo exerted during mechanical disruption to breakdown the DNA roform, HEW-lysozyme, EDTA, sonication) vary among spe polymers, the slower sedimentation rate of Solids through cific procedures reported. A variation on the HEW-lysozyme/ Viscous fluid results in poor separation of Solids and liquid 10 during centrifugation. Other than mechanical shear force, EDTA treatment using a dipolar ionic detergent in place of there exist nucleolytic enzymes that degrade DNA polymer. EDTA is discussed by Stabel et al. (1994) Veterinary Micro In E. coli, the endogenous gene endA encodes for an endo biol., 38: 307-314. For a general review of use of intracellular nuclease (molecular weight of the mature protein is approx. lytic enzyme systems to disrupt E. coli, see Dabora and 24.5 kD) that is normally secreted to the periplasm and Cooney (1990) in Advances in Biochemical Engineering/ 15 cleaves DNA into oligodeoxyribonucleotides in an endo Biotechnology, Vol. 43, A. Fiechter, ed. (Springer-Verlag: nucleolytic manner. It has been Suggested that endA is rela Berlin), pp. 11-30. tively weakly expressed by E. coli (Wackemagel et al. (1995) Conventional methods for the recovery of proteins or Gene 154: 55-59). polypeptides of interest from the cytoplasm, as soluble pro If desired, the proteins produced using one or more Strains tein or refractile particles, involved disintegration of the bac- 20 in the array of this invention may be isolated and purified to terial cell by mechanical breakage. Mechanical disruption Substantial purity by Standard techniques well known in the typically involves the generation of local cavitation in a liquid art, including, but not limited to, ammonium sulfate or etha Suspension, rapid agitation with rigid beads, Sonication, or nol precipitation, acid extraction, anion or cation exchange grinding of cell Suspension (Bacterial Cell Surface Tech chromatography, phosphocellulose chromatography, hydro niques, Hancock and Poxton (John Wiley & Sons Ltd, 1988), 25 phobic interaction chromatography, affinity chromatography, Chapter 3, p. 55). nickel chromatography, hydroxylapatite chromatography, HEW-lysozyme acts biochemically to hydrolyze the pep reverse phase chromatography, lectin chromatography, pre tidoglycan backbone of the cell wall. The method was first parative electrophoresis, detergent solubilization, selective developed by Zinder and Arndt (1956) Proc. Natl. Acad. Sci. precipitation with Such substances as column chromatogra USA, 42: 586-590, who treated E. coli with egg albumin 30 phy, immunopurification methods, and others. For example, (which contains HEW-lysozyme) to produce rounded cellular proteins having established molecular adhesion properties spheres later known as spheroplasts. These structures can be reversibly fused with a ligand. With the appropriate retained some cell-wall components but had large Surface ligand, the protein can be selectively adsorbed to a purifica areas in which the cytoplasmic membrane was exposed. U.S. tion column and then freed from the column in a relatively Pat. No. 5,169,772 discloses a method for purifying hepari- 35 pure form. The fused protein is then removed by enzymatic nase from bacteria comprising disrupting the envelope of the activity. In addition, protein can be purified using immunoaf bacteria in an osmotically-stabilized medium, e.g., 20% finity columns or Ni-NTA columns. General techniques are Sucrose solution using, e.g., EDTA, lysozyme, or an organic further described in, for example, R. Scopes, Protein Purifi compound, releasing the non-heparinase-like proteins from cation: Principles and Practice, Springer-Verlag. N.Y. (1982); the periplasmic space of the disrupted bacteria by exposing 40 Deutscher, Guide to Protein Purification, Academic Press the bacteria to a low-ionic-strength buffer, and releasing the (1990); U.S. Pat. No. 4,511,503; S. Roe, Protein Purification heparinase-like proteins by exposing the low-ionic-strength Techniques. A Practical Approach (Practical Approach washed bacteria to a buffered salt solution. Series), Oxford Press (2001); D. Bollag, et al., Protein Meth Many different modifications of these methods have been ods, Wiley-Lisa, Inc. (1996); AK Patra et al., Protein Expr used on a wide range of expression systems with varying 45 Purif, 18(2): p.182-92 (2000); and R. Mukhija, et al., Gene degrees of success (Joseph-LiaZun et al. (1990) Gene, 86: 165(2): p. 303-6 (1995). See also, for example, Ausubel, et al. 291-295; Carter et al. (1992) Bio/Technology, 10: 163-167). (1987 and periodic supplements): Deutscher (1990) “Guide Efforts to induce recombinant cell culture to produce to Protein Purification.” Methods in Enzymology Vol. 182, and lysozyme have been reported. EPO 155 189 discloses a means other volumes in this series; Coligan, etal. (1996 and periodic for inducing a recombinant cell culture to produce lysozymes, 50 Supplements) Current Protocols in Protein Science Wiley/ which would ordinarily be expected to kill such host cells by Greene, N.Y.; and manufacturer's literature on use of protein means of destroying or lysing the cell wall structure. purification products, e.g., Pharmacia, Piscataway, N.J., or U.S. Pat. No. 4,595,658 discloses a method for facilitating Bio-Rad, Richmond, Calif. Combination with recombinant externalization of proteins transported to the periplasmic techniques allow fusion to appropriate segments, e.g., to a space of bacteria. This method allows selective isolation of 55 FLAG sequence or an equivalent which can be fused via a proteins that locate in the periplasm without the need for protease-removable sequence. See also, for example, Hochuli lysozyme treatment, mechanical grinding, or osmotic shock (1989) Chemische Industrie 12:69-70; Hochuli (1990) “Puri treatment of cells. U.S. Pat. No. 4,637,980 discloses produc fication of Recombinant Proteins with Metal Chelate Absor ing a bacterial product by transforming a temperature-sensi bent in Setlow (ed.) Genetic Engineering, Principle and tive lysogen with a DNA molecule that codes, directly or 60 Methods 12:87–98, Plenum Press, NY; and Crowe, et al. indirectly, for the product, culturing the transformant under (1992) QIAexpress: The High Level Expression & Protein permissive conditions to express the gene product intracellu Purification System QUIAGEN, Inc., Chatsworth, Calif. larly, and externalizing the product by raising the temperature Detection of the expressed protein is achieved by methods to induce phage-encoded functions. Asami et al. (1997) J. known in the art and include, for example, radioimmunoas Ferment. and Bioeng., 83: 511-516 discloses synchronized 65 says, Western blotting techniques or immunoprecipitation. disruption of E. coli cells by T4 phage infection, and Tanji et Certain proteins expressed by the strains in the array of this al. (1998).J. Ferment. and Bioeng., 85: 74-78 discloses con invention may form insoluble aggregates (inclusion bod US 9,394,571 B2 33 34 ies'). Several protocols are suitable for purification of pro to column matrices and the proteins immunopurified. All of teins from inclusion bodies. For example, purification of these methods are well known in the art. It will be apparent to inclusion bodies typically involves the extraction, separation one of skill that chromatographic techniques can be per and/or purification of inclusion bodies by disruption of the formed at any scale and using equipment from many different host cells, e.g., by incubation in a buffer of 50 mMTRIS/HCL manufacturers (e.g., Pharmacia Biotech). pH 7.5, 50 mMNaCl, 5 mMMgCl, 1 mMDTT, 0.1 mMATP, Renaturation and Refolding and 1 mM PMSF. The cell suspension is typically lysed using Where heterologously expressed protein is produced in a 2-3 passages through a French Press. The cell Suspension can denatured form, insoluble protein can be renatured or also be homogenized using a Polytron (Brinkman Instru refolded to generate secondary and tertiary protein structure ments) or Sonicated on ice. Alternate methods of lysing bac 10 conformation. Protein refolding steps can be used, as neces teria are apparent to those of skill in the art (see, e.g., Sam sary, in completing configuration of the heterologous prod brook et al., Supra; Ausubel et al., Supra). uct. Refolding and renaturation can be accomplished using an If necessary, the inclusion bodies can be solubilized, and agent that is known in the art to promote dissociation/asso the lysed cell Suspension typically can be centrifuged to ciation of proteins. For example, the protein can be incubated remove unwanted insoluble matter. Proteins that formed the 15 with dithiothreitol followed by incubation with oxidized glu inclusion bodies may be renatured by dilution or dialysis with tathione disodium salt followed by incubation with a buffer a compatible buffer. Suitable solvents include, but are not containing a refolding agent such as urea. limited to urea (from about 4M to about 8 M), formamide (at The protein or polypeptide of interest can also be rena least about 80%, volume/volume basis), and guanidine tured, for example, by dialyzing it against phosphate-buffered hydrochloride (from about 4 M to about 8 M). Although saline (PBS) or 50 mMNa-acetate, pH 6 buffer plus 200 mM guanidine hydrochloride and similar agents are denaturants, NaCl. Alternatively, the protein can be refolded while immo this denaturation is not irreversible and renaturation may bilized on a column, such as the NiNTA column by using a occur upon removal (by dialysis, for example) or dilution of linear 6M-1 Murea gradient in 500 mM NaCl, 20% glycerol, the denaturant, allowing re-formation of immunologically 20 mM Tris/HCl pH 7.4, containing protease inhibitors. The and/or biologically active protein. Other suitable buffers are 25 renaturation can be performed over a period of 1.5 hours or known to those skilled in the art. more. After renaturation the proteins can be eluted by the The heterologously-expressed proteins present in the addition of 250 mMimidazole. Imidazole can be removed by Supernatant can be separated from the host proteins by stan a final dialyzing step against PBS or 50 mM sodium acetate dard separation techniques well known to those of skill in the pH 6 buffer plus 200 mM. NaCl. The purified protein can be art. For example, an initial salt fractionation can separate 30 stored at 4°C. or frozen at -80° C. many of the unwanted host cell proteins (or proteins derived Other methods include, for example, those that may be from the cell culture media) from the protein or polypeptide described in M H Lee et al., Protein Expr: Purif., 25(1): p. of interest. One Such example can be ammonium Sulfate. 166-73 (2002), W. K. Cho et al., J. Biotechnology, 77(2-3): p. Ammonium sulfate precipitates proteins by effectively reduc 169-78 (2000), Ausubel, et al. (1987 and periodic supple ing the amount of water in the protein mixture. Proteins then 35 ments), Deutscher (1990) “Guide to Protein Purification.” precipitate on the basis of their solubility. The more hydro Methods in Enzymology vol. 182, and other volumes in this phobic a protein is, the more likely it is to precipitate at lower series, Coligan, et al. (1996 and periodic Supplements) Cur ammonium sulfate concentrations. A typical protocol rent Protocols in Protein Science Wiley/Greene, N.Y., S. Roe, includes adding Saturated ammonium Sulfate to a protein Protein Purification Techniques. A Practical Approach (Prac Solution so that the resultant ammonium Sulfate concentration 40 tical Approach Series), Oxford Press (2001); D. Bollag, et al., is between 20-30%. This concentration will precipitate the Protein Methods, Wiley-Lisa, Inc. (1996) most hydrophobic of proteins. The precipitate is then dis Expression Vectors carded (unless the protein of interest is hydrophobic) and A heterologous protein of interest can be produced in one ammonium sulfate is added to the Supernatant to a concen or more of the host cells disclosed herein by introducing into tration known to precipitate the protein of interest. The pre 45 each strain an expression vector encoding the heterologous cipitate is then solubilized in buffer and the excess salt protein of interest. In one embodiment, the vector comprises removed if necessary, either through dialysis or diafiltration. a polynucleotide sequence encoding the protein of interest Other methods that rely on solubility of proteins, such as cold operably linked to a promoter capable of functioning in the ethanol precipitation, are well known to those of skill in the chosen host cell, as well as all other required transcription and art and can be used to fractionate complex protein mixtures. 50 translation regulatory elements. The molecular weight of a protein or polypeptide of inter The term “operably linked’ refers to any configuration in est can be used to isolated it from proteins of greater and which the transcriptional and any translational regulatory lesser size using ultrafiltration through membranes of differ elements are covalently attached to the encoding sequence in ent pore size (for example, Amicon or Millipore membranes). Such disposition(s), relative to the coding sequence, that in As a first step, the protein mixture can be ultrafiltered through 55 and by action of the host cell, the regulatory elements can a membrane with a pore size that has a lower molecular direct the expression of the coding sequence. weight cut-off than the molecular weight of the protein of The heterologous protein of interest can be expressed from interest. The retentate of the ultrafiltration can then be ultra polynucleotides in which the heterologous polypeptide cod filtered against a membrane with a molecular cut off greater ing sequence is operably linked to transcription and transla than the molecular weight of the protein of interest. The 60 tion regulatory elements to form a functional gene from protein or polypeptide of interest will pass through the mem which the host cell can express the protein or polypeptide. brane into the filtrate. The filtrate can then be chromato The coding sequence can be a native coding sequence for the graphed as described below. heterologous polypeptide, or may be a coding sequence that The expressed proteins or polypeptides of interest can also has been selected, improved, or optimized for use in the be separated from other proteins on the basis of its size, net 65 selected expression host cell: for example, by synthesizing Surface charge, hydrophobicity, and affinity for ligands. In the gene to reflect the codon use bias of a host species. In one addition, antibodies raised against proteins can be conjugated embodiment of the invention, the host species is a Pfluore US 9,394,571 B2 35 36 scens, and the codon bias of P. fluorescens is taken into and the signal sequence capable of directing compartmental account when designing the polypeptide coding sequence. accumulation or secretion of the translated protein. Option The gene(s) are constructed within or inserted into one or ally the heterologous sequence can encode a fusion enzyme more vector(s), which can then be transformed into the including an N-terminal identification polypeptide imparting expression host cell. desired characteristics, e.g., stabilization or simplified puri Other regulatory elements may be included in a vector fication of expressed heterologous product. The fusion (also termed “expression construct”). The vector will typi polypeptide can also comprise one or more target proteins or cally comprise one or more phenotypic selectable markers inhibitors or enhances thereof, as discussed Supra. and an origin of replication to ensure maintenance of the Vectors are known in the art for expressing heterologous vector and to, if desirable, provide amplification within the 10 host. Additional elements include, but are not limited to, for proteins in host cells, and any of these may be used for example, transcriptional enhancer sequences, translational expressing the genes according to the present invention. Such enhancer sequences, other promoters, activators, transla vectors include, e.g., plasmids, cosmids, and phage expres tional start and stop signals, transcription terminators, cis sion vectors. Examples of useful plasmid vectors include, but tronic regulators, polycistronic regulators, or tag sequences, 15 are not limited to, the expression plasmids pBBR1MCS, Such as nucleotide sequence "tags' and “tag” polypeptide pDSK519, pKT240, pML122, pPS10, RK2, RK6, pRO1600, coding sequences, which facilitates identification, separation, and RSF 1010. Other examples of such useful vectors include purification, and/or isolation of an expressed polypeptide. those described by, e.g.: N. Hayase, in Appl. Envir. Microbiol. In another embodiment, the expression vector further com 60(9):3336-42 (September 1994); A. A. Lushnikov et al., in prises a tag sequence adjacent to the coding sequence for the Basic Life Sci. 30:657-62 (1985): S. Graupner & W. Wack protein or polypeptide of interest. In one embodiment, this tag emagel, in Biomolec.Eng. 17(1):11-16. (October 2000); H. P. sequence allows for purification of the protein. The tag Schweizer, in Curr. Opin. Biotech. 12(5):439-45 (October sequence can be an affinity tag, Such as a hexa-histidine 2001); M. Bagdasarian & K. N. Timmis, in Curr. Topics affinity tag. In another embodiment, the affinity tag can be a Microbiol. Immunol.96:47-67 (1982); T. Ishiiet al., in FEMS glutathione-S-transferase molecule. The tag can also be a 25 Microbiol. Lett. 116(3):307-13 (Mar. 1, 1994); I. N. Olekh fluorescent molecule, such as YFP or GFP, or analogs of such novich & Y. K. Fomichev, in Gene 140(1):63-65 (Mar. 11, fluorescent proteins. The tag can also be a portion of an 1994); M. Tsuda & T. Nakazawa, in Gene 136(1-2):257-62 antibody molecule, or a known antigen or ligand for a known (Dec. 22, 1993); C. Nieto et al., in Gene 87(1): 145-49 (Mar. 1, binding partner useful for purification. 1990); J. D. Jones & N. Gutterson, in Gene 61(3):299-306 A protein-encoding gene according to the present inven 30 (1987); M. Bagdasarian et al., in Gene 16(1-3):237-47 (De tion can include, in addition to the protein coding sequence, cember 1981); H. P. Schweizer et al., in Genet. Eng. (NY) the following regulatory elements operably linked thereto: a 23:69-81 (2001): P. Mukhopadhyay et al., in J. Bact. 172(1): promoter, a ribosome binding site (RBS), a transcription ter 477-80 (January 1990); D.O. Wood et al., in J. Bact. 145(3): minator, and translational start and stop signals. Useful RBSS 1448-51 (March 1981); and R. Holtwicket al., in Microbiol can be obtained from any of the species useful as host cells in 35 ogy 147(Pt 2):337-44 (February 2001). expression systems according to the present invention, pref Further examples of expression vectors that can be useful erably from the selected host cell. Many specific and a variety in a host cell of the invention include those listed in Table 5 as of consensus RBSS are known, e.g., those described in and derived from the indicated replicons. referenced by D. Frishman et al., Gene 234(2):257-65 (8 Jul. 1999); and B. E. Suzek et al., Bioinformatics 17(12): 1123-30 40 TABLE 5 (December 2001). In addition, either native or synthetic RBSs Examples of Useful Expression Vectors may be used, e.g., those described in: EP 0207459 (synthetic RBSs); O. Ikehata et al., Eur. J. Biochem. 181(3):563-70 Replicon Vector(s) (1989) (native RBS sequence of 5'-AAGGAAG-3'). Further PPS10 PCN39, PCN51 examples of methods, vectors, translation and transcription 45 RSF1010 PKT261-3 elements, and other elements useful in the present invention PMMB66EH PEB8 are described in, e.g.: U.S. Pat. No. 5,055.294 to Gilroy and PPLGN1 U.S. Pat. No. 5,128,130 to Gilroy et al.; U.S. Pat. No. 5,281, PMYC1050 532 to Rammler et al.; U.S. Pat. Nos. 4,695,455 and 4,861, RK2 RP1 PRK415 595 to Barnes et al.; U.S. Pat. No. 4,755,465 to Gray et al.; and 50 PJB653 U.S. Pat. No. 5,169,760 to Wilcox. PRO1600 PUCP Transcription of the DNA encoding the heterologous pro PBSP tein of interest is increased by inserting an enhancer sequence into the vector or plasmid. Typical enhancers are cis-acting The expression plasmid, RSF1010, is described, e.g., by F. elements of DNA, usually about from 10 to 300 bp in size that 55 Heffron et al., in Proc. Natl Acad. Sci. USA 72(9):3623-27 act on the promoter to increase its transcription. Examples (September 1975), and by K. Nagahari & K. Sakaguchi, in J. include various Pseudomonas enhancers. Bact. 133(3):1527-29 (March 1978). Plasmid RSF 1010 and Generally, the heterologous expression vectors will derivatives thereof are particularly useful vectors in the include origins of replication and selectable markers permit present invention. Exemplary useful derivatives of RSF1010, ting transformation of the host cell and a promoter derived 60 which are known in the art, include, e.g., pKT212, pKT214, from a highly-expressed gene to direct transcription of a pKT231 and related plasmids, and pMYC1050 and related downstream structural sequence. Such promoters can be plasmids (see, e.g., U.S. Pat. Nos. 5,527,883 and 5,840,554 to derived from operons encoding the enzymes such as 3-phos Thompson et al.), such as, e.g., pMYC1803. Plasmid phoglycerate kinase (PGK), acid phosphatase, or heat shock pMYC1803 is derived from the RSF1010-based plasmid proteins, among others. Where signal sequences are used, the 65 pTJS260 (see U.S. Pat. No. 5,169,760 to Wilcox), which heterologous coding sequence is assembled in appropriate carries a regulated tetracycline resistance marker and the phase with translation initiation and termination sequences, replication and mobilization loci from the RSF 1010 plasmid. US 9,394,571 B2 37 38 Other exemplary useful vectors include those described in tein; AraC family transcriptional activators; repressor pro U.S. Pat. No. 4,680,264 to Puhler et al. teins, e.g., E. coli LacI proteins; and dual-function regulatory In one embodiment, an expression plasmid is used as the proteins, e.g., E. coli NagO protein. Many regulated-pro expression vector. In another embodiment, RSF1010 or a moter/promoter-regulatory-protein pairs are known in the art. derivative thereof is used as the expression vector. In still In one embodiment, the expression construct for the target another embodiment, pMYC1050 or a derivative thereof, or protein(s) and the heterologous protein of interest are under pMYC4803 or a derivative thereof, is used as the expression the control of the same regulatory element. Vector. Promoter regulatory proteins interact with an effector com The plasmid can be maintained in the host cell by inclusion pound, i.e. a compound that reversibly or irreversibly associ of a selection marker gene in the plasmid. This may be an 10 ates with the regulatory protein so as to enable the protein to antibiotic resistance gene(s), where the corresponding anti either release or bind to at least one DNA transcription regu biotic(s) is added to the fermentation medium, or any other latory region of the gene that is under the control of the type of selection marker gene known in the art, e.g., a pro promoter, thereby permitting or blocking the action of a tran totrophy-restoring gene where the plasmid is used in a host 15 Scriptase enzyme in initiating transcription of the gene. Effec cell that is auxotrophic for the corresponding trait, e.g., a tor compounds are classified as either inducers or co-repres biocatalytic trait such as an amino acid biosynthesis or a sors, and these compounds include native effector nucleotide biosynthesis trait, or a carbon Source utilization compounds and gratuitous inducer compounds. Many regu trait. lated-promoter/promoter-regulatory-protein/effector-com The promoters used in accordance with the present inven pound trios are known in the art. Although an effector com tion may be constitutive promoters or regulated promoters. pound can be used throughout the cell culture or Common examples of useful regulated promoters include those of the family derived from the lac promoter (i.e. the lacZ fermentation, in a preferred embodiment in which a regulated promoter), especially the tac and trc promoters described in promoter is used, after growth of a desired quantity or density U.S. Pat. No. 4,551,433 to DeBoer, as well as Ptac16, Ptac17, of host cell biomass, an appropriate effector compound is PtacII, PlacUV5, and the T71ac promoter. In one embodi 25 added to the culture to directly or indirectly result in expres ment, the promoter is not derived from the host cell organism. sion of the desired gene(s) encoding the protein or polypep In certain embodiments, the promoter is derived from an E. tide of interest. coli organism. By way of example, where a lac family promoter is uti Common examples of non-lac-type promoters useful in lized, a lacI gene can also be present in the system. The lacI expression systems according to the present invention 30 gene, which is (normally) a constitutively expressed gene, include, e.g., those listed in Table 6. encodes the Lac repressor protein (LacD protein) which binds to the lac operator of these promoters. Thus, where a lac TABLE 6 family promoter is utilized, the lacI gene can also be included and expressed in the expression system. In the case of the lac Examples of non-lac Promoters 35 promoter family members, e.g., the tac promoter, the effector Promoter Inducer compound is an inducer, preferably a gratuitous inducer Such as IPTG (isopropyl-D-1-thiogalactopyranoside, also called PR High temperature P. High temperature "isopropylthiogalactoside'). Pn Alkyl- or halo-benzoates 40 For expression of a protein or polypeptide of interest, any Pl Alkyl- or halo-toluenes plant promoter may also be used. A promoter may be a plant Psal Salicylates RNA polymerase II promoter. Elements included in plant promoters can be a TATA box or Goldberg-Hogness box, See, e.g.: J. Sanchez-Romero & V. De Lorenzo (1999) typically positioned approximately 25 to 35 basepairs Manual of Industrial Microbiology and Biotechnology (A. 45 upstream (5') of the transcription initiation site, and the Demain & J. Davies, eds.) pp. 460-74 (ASM Press, Washing CCAAT box, located between 70 and 100 basepairs ton, D.C.); H. Schweizer (2001) Current Opinion in Biotech upstream. In plants, the CCAAT box may have a different nology, 12:439-445; and R. Slater & R. Williams (2000 consensus sequence than the functionally analogous Molecular Biology and Biotechnology (J. Walker & R. Rap sequence of mammalian promoters (Messing et al. (1983) In: ley, eds.) pp. 125-54 (The Royal Society of Chemistry, Cam 50 Genetic Engineering of Plants, Kosuge et al., eds., pp. 211 bridge, UK)). A promoter having the nucleotide sequence of 227). In addition, virtually all promoters include additional a promoter native to the selected bacterial host cell may also upstream activating sequences or enhancers (Benoist and be used to control expression of the transgene encoding the Chambon (1981) Nature 290:304-310: Gruss et al. (1981) target polypeptide, e.g., a Pseudomonas anthranilate or ben Proc. Nat. Acad. Sci. 78:943-947; and Khoury and Gruss Zoate operon promoter (Pant, Pben). Tandem promoters may 55 (1983) Cell 27:313-314) extending from around -100 bp to also be used in which more than one promoter is covalently -1,000 bp or more upstream of the transcription initiation attached to another, whether the same or different in site. sequence, e.g., a Pant-Pben tandem promoter (interpromoter Expression Systems hybrid) or a Plac-Plac tandem promoter, or whether derived It may be desirable to target the protein or polypeptide of from the same or different organisms. 60 interest to the periplasm of one or more of the populations of Regulated promoters utilize promoter regulatory proteins host cells in the array, or into the extracellular space. In one in order to control transcription of the gene of which the embodiment, the expression vector further comprises a nucle promoter is a part. Where a regulated promoter is used herein, otide sequence encoding a secretion signal sequence a corresponding promoter regulatory protein will also be part polypeptide operably linked to the nucleotide sequence of an expression system according to the present invention. 65 encoding the protein or polypeptide of interest. In some Examples of promoter regulatory proteins include: activator embodiments, no modifications are made between the signal proteins, e.g., E. coli catabolite activator protein, MalT pro sequence and the protein or polypeptide of interest. However, US 9,394,571 B2 39 40 in certain embodiments, additional cleavage signals are incor protoplast fusion, bacterial conjugation, and divalent cation porated to promote proper processing of the amino terminal treatment, e.g., calcium chloride treatment or CaCl/Mg2+ of the polypeptide. treatment, or other well known methods in the art. See, e.g., The vector can have any of the characteristics described Morrison, J. Bact., 132:349-351 (1977); Clark-Curtiss & above. In one embodiment, the vector comprising the coding Curtiss, Methods in Enzymology, 101:347-362 (Wu et al., eds. sequence for the protein or polypeptide of interest further 1983), Sambrook et al., Molecular Cloning, A Laboratory comprises a signal sequence, e.g., a secretion signal Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expres Sequence. Sion: A Laboratory Manual (1990); and Current Protocols in Therefore, in one embodiment, this isolated polypeptide is Molecular Biology (Ausubel et al., eds., 1994)). a fusion protein of the secretion signal and a protein or 10 Proteins of Interest polypeptide of interest. However, the secretion signal can also The methods and compositions of the present invention are be cleaved from the protein when the protein is targeted to the useful for identifying a Pfluorescens strain that is optimal for periplasm. In one embodiment, the linkage between the Sec producing high levels of a properly processed protein or system secretion signal and the protein or polypeptide is polypeptide of interest. The arrays are useful for screening for modified to increase cleavage of the secretion signal. 15 production of a protein or polypeptide of interest of any The CHAMPIONTM pET expression system provides a species and of any size. However, in certain embodiments, the high level of protein production. Expression is induced from protein or polypeptide of interest is a therapeutically useful the strong T7lac promoter. This system takes advantage of the protein or polypeptide. In some embodiments, the protein can high activity and specificity of the bacteriophage T7 RNA be a mammalian protein, for example a human protein, and polymerase for high level transcription of the gene of interest. can be, for example, a growth factor, a cytokine, a chemokine The lac operator located in the promoter region provides or a blood protein. The protein or polypeptide of interest can tighter regulation than traditional T7-based vectors, improv be processed in a similar manner to the native protein or ing plasmid stability and cell viability (Studier and Moffatt polypeptide. In certain embodiments, the protein or polypep (1986).J Molecular Biology 189(1): 113-30; Rosenberg, et al. tide of interest is less than 100kD, less than 50kD, or less than (1987) Gene 56(1): 125-35). The T7 expression system uses 25 30kD in size. In certain embodiments, the protein or polypep the T7 promoter and T7 RNA polymerase (T7 RNAP) for tide of interest is a polypeptide of at least about 5, 10, 15, 20, high-level transcription of the gene of interest. High-level 30, 40, 50 or 100 or more amino acids. expression is achieved in T7 expression systems because the The coding sequence for the protein or polypeptide of T7 RNAP is more processive than native E. coli RNAP and is interest can be a native coding sequence for the polypeptide, dedicated to the transcription of the gene of interest. Expres 30 if available, but will more preferably be a coding sequence sion of the identified gene is induced by providing a source of that has been selected, improved, or optimized for use in an T7 RNAP in the host cell. This is accomplished by using a expressible form in the strains of the array: for example, by BL21 E. coli host containing a chromosomal copy of the T7 optimizing the gene to reflect the codon use bias of a RNAP gene. The T7 RNAP gene is under the control of the Pseudomonas species such as Pfluorescens or other Suitable lacUV5 promoter which can be induced by IPTG. T7 RNAP 35 organism. For gene optimization, one or more rare codons is expressed upon induction and transcribes the gene of inter may be removed to avoid ribosomal stalling and minimize eSt. amino acid misincorporation. One or more gene-internal The pBAD expression system allows tightly controlled, ribosome binding sites may also be eliminated to avoid trun titratable expression of protein or polypeptide of interest cated protein products. Long stretches of Cand G nucleotides through the presence of specific carbon Sources such as glu 40 may be removed to avoid RNA polymerase slippage that cose, glycerol and arabinose (Guzman, et al. (1995) J Bacte could result in frame-shifts. Strong gene-internal stem-loop riology 177(14): 4121-30). The pBAD vectors are uniquely structures, especially the ones covering the ribosome binding designed to give precise control over expression levels. Het site, may also be eliminated. erologous gene expression from the pBAD Vectors is initiated In other embodiments, the protein when produced also at the araBAD promoter. The promoter is both positively and 45 includes an additional targeting sequence, for example a negatively regulated by the product of the araC gene. AraC is sequence that targets the protein to the periplasm or the extra a transcriptional regulator that forms a complex with L-ara cellular medium. In one embodiment, the additional targeting binose. In the absence of L-arabinose, the AraC dimer blocks sequence is operably linked to the carboxy-terminus of the transcription. For maximum transcriptional activation two protein. In another embodiment, the protein includes a secre events are required: (i) L-arabinose binds to AraC allowing 50 tion signal for an autotransporter, a two partner secretion transcription to begin, and, (ii) The cAMP activator protein system, a main terminal branch system or a fimbrial usher (CAP)-cAMP complex binds to the DNA and stimulates porin. binding of AraC to the correct location of the promoter The gene(s) that result are constructed within or are region. inserted into one or more vectors, and then transformed into The trc expression system allows high-level, regulated 55 each of the host cell populations in the array. Nucleic acid or expression in E. coli from the trc promoter. The trc expression a polynucleotide said to be provided in an “expressible form’ vectors have been optimized for expression of eukaryotic means nucleic acid or a polynucleotide that contains at least genes in E. coli. The trc promoter is a strong hybrid promoter one gene that can be expressed by the one or more of the host derived from the tryptophane (trp) and lactose (lac) promot cell populations of the invention. ers. It is regulated by the lacO operator and the product of the 60 Extensive sequence information required for molecular lacIQ gene (Brosius, J. (1984) Gene 27(2):161-72). genetics and genetic engineering techniques is widely pub Transformation of the host cells with the vector(s) dis licly available. Access to complete nucleotide sequences of closed herein may be performed using any transformation mammalian, as well as human, genes, cDNA sequences, methodology known in the art, and the bacterial host cells amino acid sequences and genomes can be obtained from may be transformed as intact cells or as protoplasts (i.e. 65 GenBank. GenBank is maintained by the National Institutes including cytoplasts). Exemplary transformation methodolo of Health, Bethesda, Md., and can be accessed at ncbi.nlm gies include poration methodologies, e.g., electroporation, .nih.gov/Entrez, within the NIH website. Additional informa US 9,394,571 B2 41 42 tion can also be obtained from GeneCards, an electronic photoxin), nerve growth factors (e.g., NGF), vascular endot encyclopedia integrating information about genes and their helial growth factor (VEGF); interferons (e.g., IFN-O, IFN-B, products and biomedical applications, made available by the IFN-Y); leukemia inhibitory factor (LIF); ciliary neurotrophic Department of Molecular Genetics, the Weizmann Institute factor (CNTF); oncostatin M: stem cell factor (SCF); trans of Science, Rehovot, Israel. Nucleotide sequence information forming growth factors (e.g., TGF-C. TGF-B1, TGF-32, also can be obtained from the EMBL Nucleotide Sequence TGF-B3); TNF superfamily (e.g., LIGHT/TNFSF14, Database made available on the worldwide web by the Euro STALL-1/TNFSF13B (BLyS, BAFF, THANK), TNFalpha/ pean Bioinformatics Institute (Hinxton, Cambridge, UK) or TNFSF2 and TWEAK/TNFSF12); or chemokines (BCA-1/ from the DNA Databank of Japan (Research Organization of BLC-1, BRAK/Kec, CXCL16, CXCR3. ENA-78/LIX, Information and Systems, National Institute of Genetics, 10 Eotaxin-1, Eotaxin-2/MPIF-2, Exodus-2/SLC, Fractalkine? Center for Information Biology and DNA Data Bank of Neurotactin, GROalpha/MGSA, HCC-1, I-TAC, Lymphotac Japan, 1111 Yata, Mishima, Shizuoka 411-8540, Japan). tin/ATAC/SCM, MCP-1/MCAF, MCP-3, MCP-4, MDC/ Additional sites for information on amino acid sequences STCP-1/ABCD-1, MIP-1...quadrature. MIP-1.cquadrature., include the Protein Information Resource website established MIP-2.quadrature./GRO.cquadrature. MIP-3.quadrature./ by the National Biomedical Research Foundation, which 15 Exodus/LARC, MIP-3/Exodus-3/ELC, MIP-4/PARC/DC includes Swiss-Prot. CK1, PF-4. RANTES, SDF1, TARC, TECK, microbial tox Examples of proteins that can be expressed in this inven ins, ADP ribosylating toxins, microbial or viral antigens). tion include molecules Such as, e.g., renin, a growth hormone, In one embodiment of the present invention, the protein of including human growth hormone; bovine growth hormone; interest can be a multi-subunit protein or polypeptide. Mul growth hormone releasing factor, parathyroid hormone; thy tisubunit proteins that can be expressed include homomeric roid stimulating hormone; lipoproteins; C-1-antitrypsin; and heteromeric proteins. The multisubunit proteins may insulin A-chain; insulin B-chain; proinsulin; thrombopoietin: include two or more subunits that may be the same or differ follicle stimulating hormone; calcitonin; luteinizing hor ent. For example, the protein may be a homomeric protein mone; glucagon; clotting factors such as factor VIIIC, factor comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more subunits. IX, tissue factor, and von Willebrands factor; anti-clotting 25 The protein also may be a heteromeric protein including 2, 3, factors such as Protein C. atrial naturietic factor; lung Surfac 4, 5, 6, 7, 8, 9, 10, 11, 12, or more subunits. Exemplary tant; a plasminogen activator, Such as urokinase or human multisubunit proteins include: receptors including ion chan urine or tissue-type plasminogen activator (t-PA); bombesin; nel receptors; extracellular matrix proteins including chon thrombin; hemopoietic growth factor, tumor necrosis factor droitin; collagen; immunomodulators including MHC pro alpha and -beta; enkephalinase; a serum albumin Such as 30 teins, full chain antibodies, and antibody fragments; enzymes human serum albumin; mullerian-inhibiting Substance; including RNA polymerases, and DNA polymerases; and relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadot membrane proteins. ropin-associated polypeptide; a microbial protein, such as In another embodiment, the protein of interest can be a beta-lactamase; Dnase; inhibin; activin; vascular endothelial blood protein. The blood proteins expressed in this embodi growth factor (VEGF); receptors for hormones or growth 35 ment include but are not limited to carrier proteins, such as factors; integrin: protein A or D; rheumatoid factors; a neu albumin, including human and bovine albumin, transferrin, rotrophic factor such as brain-derived neurotrophic factor recombinant transferrin half-molecules, haptoglobin, (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or fibrinogen and other coagulation factors, complement com NT-6), or a nerve growth factor such as NGF-?3; cardiotro ponents, immunoglobulins, enzyme inhibitors, precursors of phins (cardiac hypertrophy factor) Such as cardiotrophin-1 40 Substances such as angiotensin and bradykinin, insulin, (CT-1); platelet-derived growth factor (PDGF): fibroblast endothelin, and globulin, including alpha, beta, and gamma growth factor such as aFGF and bFGF; epidermal growth globulin, and other types of proteins, polypeptides, and frag factor (EGF); transforming growth factor (TGF) such as ments thereof found primarily in the blood of mammals. The TGF-alpha and TGF-B, including TGF-?31, TGF-B2, TGF amino acid sequences for numerous blood proteins have been B3, TGF-B4, or TGF-35; insulin-like growth factor-I and -II 45 reported (see, S. S. Baldwin (1993) Comp. Biochem Physiol. (IGF-I and IGF-II): des(1-3)-IGF-I (brain IGF-I), insulin-like 106b:203-218), including the amino acid sequence for human growth factor binding proteins; CD proteins such as CD-3, serum albumin (Lawn, L. M., et al. (1981) Nucleic Acids CD-4, CD-8, and CD-19; erythropoietin; osteoinductive fac Research, 9:6103–61 14.) and human serum transferrin (Yang, tors; immunotoxins; a bone morphogenetic protein (BMP); F. et al. (1984) Proc. Natl. Acad. Sci. USA 81:2752-2756). an interferon Such as interferon-alpha, -beta, and -gamma; 50 In another embodiment, the protein of interest can be an colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, enzyme or co-factor. The enzymes and co-factors expressed and G-CSF; interleukins (ILS), e.g., IL-1 to IL-10; anti in this embodiment include but are not limited to aldolases, HER-2 antibody; superoxide dismutase; T-cell receptors; sur amine oxidases, amino acid oxidases, aspartases, B12 depen face membrane proteins; decay accelerating factor, viral anti dent enzymes, carboxypeptidases, carboxyesterases, car gen Such as, for example, a portion of the AIDS envelope; 55 boxylyases, chemotrypsin, CoA requiring enzymes, cyano transport proteins; homing receptors; addressins; regulatory hydrin synthetases, cystathione synthases, decarboxylases, proteins; antibodies; and fragments of any of the above-listed dehydrogenases, alcohol dehydrogenases, dehydratases, dia polypeptides. phorases, dioxygenases, enoate reductases, epoxide In certain embodiments, the protein or polypeptide can be hydrases, fumerases, galactose oxidases, glucose isomerases, selected from IL-1, IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, 60 glucose oxidases, glycosyltrasferases, methyltransferases, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-12elasti, IL-13, nitrile hydrases, nucleoside phosphorylases, oxidoreduc IL-15, IL-16, IL-18, IL-18BPa, IL-23, IL-24, VIP, erythro tases, oxynitilases, peptidases, glycosyltrasferases, peroxi poietin, GM-CSF, G-CSF, M-CSF, platelet derived growth dases, enzymes fused to a therapeutically active polypeptide, factor (PDGF), MSF, FLT-3 ligand, EGF, fibroblast growth tissue plasminogen activator; urokinase, reptilase, streptoki factor (FGF; e.g., C-FGF (FGF-1), B-FGF (FGF-2), FGF-3, 65 nase; catalase, Superoxide dismutase; Dnase, amino acid FGF-4, FGF-5, FGF-6, or FGF-7), insulin-like growth factors hydrolases (e.g., asparaginase, amidohydrolases); carbox (e.g., IGF-1, IGF-2); tumor necrosis factors (e.g., TNF, Lym ypeptidases; proteases, trypsin, pepsin, chymotrypsin, US 9,394,571 B2 43 44 papain, bromelain, collagenase; neuramimidase; lactase, 13525); Pseudomonas fluorescens biotype B, also called bio maltase, Sucrase, and arabinofuranosidases. var 2 or biovar II (ATCC 17816); Pseudomonas fluorescens In another embodiment, the protein of interest can be a biotype C, also called biovar 3 or biovar III (ATCC 17400); single chain, Fab fragment and/or full chain antibody or frag Pseudomonas fluorescens biotype F, also called biovar 4 or ments or portions thereof. A single-chain antibody can biovar IV (ATCC 12983); Pseudomonas fluorescens biotype include the antigen-binding regions of antibodies on a single G, also called biovar 5 or biovar V (ATCC 17518); Pseudomo stably-folded polypeptide chain. Fab fragments can be apiece nas fluorescens biovar VI; Pseudomonas fluorescens Pf()-1; of a particular antibody. The Fab fragment can contain the Pseudomonas fluorescens Pf-5 (ATCC BAA-477); antigen binding site. The Fab fragment can contain 2 chains: Pseudomonas fluorescens SBW25; and Pseudomonas fluo a light chainanda heavy chain fragment. These fragments can 10 rescens subsp. cellulosa (NCIMB 10462). be linked via a linker or a disulfide bond. The host cell can be selected from “Gram-negative Proteo In other embodiments, the protein of interest is a protein bacteria Subgroup 19.” “Gram-negative Proteobacteria Sub that is active at a temperature from about 20 to about 42°C. In group 19 is defined as the group of all strains of Pseudomo one embodiment, the protein is active at physiological tem nas fluorescens biotype A. A particularly preferred strain of peratures and is inactivated when heated to high or extreme 15 this biotype is P. fluorescens strain MB101 (see U.S. Pat. No. temperatures, such as temperatures over 65° C. 5,169,760 to Wilcox), and derivatives thereof. An example of In one embodiment, the protein of interest is a protein that a preferred derivative thereof is Pfluorescens strain MB214, is active at a temperature from about 20 to about 42°C., constructed by inserting into the MB101 chromosomal asd and/or is inactivated when heated to high or extreme tempera (aspartate dehydrogenase gene) locus, a native E. coli PlacI tures, such as temperatures over 65° C.; is, or is substantially lacI-laczYA construct (i.e. in which PlacZ was deleted). homologous to, a native protein, such as a native mammalian Additional Pfluorescens strains that can be used in the or human protein and not expressed from nucleic acids in present invention include Pseudomonas fluorescens Migula concatameric form, where the promoter is not a native pro and Pseudomonas fluorescens Loitokitok, having the follow moter in to the host cell used in the array but is derived from ing ATCC designations: NCIB 8286; NRRL B-1244: NCIB another organism, Such as E. coli. 25 8865 strain CO1, NCIB 8866 strain CO; 1291 ATCC Host Cell 17458; IFO 15837: NCIB 8917; LA; NRRL B-1864; pyrro In one embodiment the invention provides an array of P lidine: PW2 ICMP 3966; NCPPB 967; NRRL B-899); fluorescens host cells from which to optimally produce a 13475; NCTC 10038; NRRL B-1603 6; IFO 15840:52-1C: heterologous protein or peptide of interest. Pfluorescens has CCEB 488-A BU 140); CCEB 553 EM15/47: IAM 1008 been demonstrated to be an improved platform for production 30 AHH-27; IAM 1055 AHH-23; 1 (IFO 15842; 12 ATCC of a variety of proteins and several efficient secretion signals 25323; NIH 11; den Dooren de Jong 216; 18 IFO 15833; have been identified from this organism (see, U.S. Applica WRRLP-7: 93 TR-10; 108 52-22; IFO 15832; 143 IFO tion Publication Number 2006/0008877, herein incorporated 15836; PL: 1492-40-40; IFO 15838; 182 IFO 3081; PJ by reference in its entirety). 73; 184IFO 15830; 185 W2L-1; 186IFO 15829; PJ 79; The Pseudomonads system offers advantages for commer 35 187 NCPPB 263; 188 NCPPB 3.16; 189 PJ227; 1208): cial expression of polypeptides and enzymes, in comparison 191 (IFO 15834; PJ 236; 22/1; 194 Klinge R-60; PJ 253); with other bacterial expression systems. In particular, Pfluo 196 PJ 288: 197 PJ 290; 198 PJ 302; 201 PJ 368; 202 rescens has been identified as an advantageous expression PJ 372; 203 PJ 376; 204 IFO 15835; PJ 682): 205 PJ system. Pfluorescens encompasses a group of common, non 686: 206PJ 692; 207 PJ 693): 208PJ 722; 212. PJ 832); pathogenic saprophytes that colonize soil, water and plant 40 215 PJ 849: 216 PJ 885; 267 B-9: 271 B-1612; 401 surface environments. Commercial enzymes derived from P C71A; IFO 15831; PJ 187; NRRL B-3 1784; IFO. 15841; fluorescens have been used to reduce environmental contami KY 8521:3081: 30-21; IFO3081; N: PYR; PW; D946-B83 nation, as detergent additives, and for Stereoselective BU 2183: FERM-P 3328; P-2563 FERM-P 2894; IFO hydrolysis. Pfluorescens is also used agriculturally to control 13658); IAM-112643F: M-1: A506 A5-06); A505 A5 pathogens. U.S. Pat. No. 4,695,462 describes the expression 45 05-1: A526 A5-26: B69; 72; NRRL B-4290; PMW6 of recombinant bacterial proteins in Pfluorescens. NCIB 11615: SC 12936; A1 IFO 15839); F 1847 CDC However, it is contemplated that alternate host cells, or EB; F 1848 CDC93): NCIB 10586; P17; F-12: AmMS 257; even a multiplicity of different host cells, can be used to PRA25; 6133D02: 6519E01; Ni; SC15208; BNL-WVC: generate an array comprising a plurality of phenotypically NCTC 2583 NCIB 81.94: H13; 1013 ATCC 11251; CCEB distinct host cells that have been genetically modified to 50 295]: IFO 3903: 1062; or Pf-5. modulate the expression of one or more target genes, as In one embodiment, the host cell can be any cell capable of discussed Supra. The host cell can be any organism in which producing a protein or polypeptide of interest, including a P target genes can be altered. Methods of identifying target fluorescens cell as described above. The most commonly used genes homologous to those listed in Tables 1 and 2 are known systems to produce proteins or polypeptides of interest in the art. Further, one of skill in the art would understand how 55 include certain bacterial cells, particularly E. coli, because of to identify target genes that are native to or useful in a host cell their relatively inexpensive growth requirements and poten of interest. Many of these proteins are well known in the art. tial capacity to produce protein in large batch cultures. Yeasts See, for example, U.S. Patent Application Publication No. are also used to express biologically relevant proteins and 2006/0110747). polypeptides, particularly for research purposes. Systems Host cells can be selected from “Gram-negative Proteobac 60 include Saccharomyces cerevisiae or Pichia pastoris. These teria Subgroup 18.” “Gram-negative Proteobacteria Sub systems are well characterized, provide generally acceptable group 18' is defined as the group of all Subspecies, varieties, levels of total protein production and are comparatively fast strains, and other Sub-special units of the species Pseudomo and inexpensive. Insect cell expression systems have also nas fluorescens, including those belonging, e.g., to the fol emerged as an alternative for expressing recombinant pro lowing (with the ATCC or other deposit numbers of exem 65 teins in biologically active form. In some cases, correctly plary strain(s) shown in parenthesis): Pseudomonas folded proteins that are post-translationally modified can be fluorescens biotype A, also called biovar 1 or biovar I (ATCC produced. Mammalian cell expression systems, such as Chi US 9,394,571 B2 45 46 nese hamster ovary cells, have also been used for the expres TABLE 7 sion of proteins or polypeptides of interest. On a small scale, these expression systems are often effective. Certain biolog Families and Genera Listed in the Part, “Gram-Negative Aerobic Rods and ics can be derived from proteins, particularly in animal or Cocci' (in Bergey (1974)) human health applications. In another embodiment, the host Family I. Pseudomonaceae Giuconobacter cell is a plant cell, including, but not limited to, a tobacco cell, Pseudomonas Xanthomonas corn, a cell from an Arabidopsis species, potato or rice cell. Zoogloea In another embodiment, the host cell can be a prokaryotic Family II. Azotobacteraceae Azomonas cell Such as a bacterial cell including, but not limited to, an Azotobacter Escherichia or a Pseudomonas species. Typical bacterial cells 10 Beijerinckia Dexia are described, for example, in “Biological Diversity: Bacteria Family III. Rhizobiaceae Agrobacterium and Archaeans, a chapter of the On-Line Biology Book, Rhizobium provided by Dr. M.J. Farabee of the Estrella Mountain Com Family IV. Methylomonadaceae Methylococcus munity College, Arizona, USA. In certain embodiments, the Methylomonas 15 Family V. Halobacteriaceae Haiobacterium host cell can be a Pseudomonad cell, and can typically be a P. Haiococcus fluorescens cell. In other embodiments, the host cell can also Other Genera Acetobacter be an E. coli cell. In another embodiment the host cell can be Alcaligenes a eukaryotic cell, for example an insect cell, including but not Bordeteia Bruceiia limited to a cell from a Spodoptera, Trichoplusia, Drosophila Franciselia or an Estigmene species, or a mammalian cell, including but Thermits not limited to a murine cell, a hamster cell, a monkey cell, a primate cell or a human cell. In one embodiment, the host cell can be a member of any of “Gram-negative Proteobacteria Subgroup 1 also includes the bacterial taxa. The cell can, for example, be a member of Proteobacteria that would be classified in this heading any species of eubacteria. The host can be a member of any 25 according to the criteria used in the classification. The head one of the taxa: Acidobacteria, Actinobacteira, Aquificae, ing also includes groups that were previously classified in this Bacteroidetes, Chlorobi, Chlamydiae, Choroflexi, Chrysio section but are no longer, Such as the genera Acidovorax, genetes, Cyanobacteria, Deferribacteres, Deinococcus, Dic Brevundimonas, Burkholderia, Hydrogenophaga, Oceani tyoglomi, Fibrobacteres, Firmicutes, Fusobacteria, Gemma monas, Ralstonia, and Stenotrophomonas, the genus Sphin timonadetes, Lentisphaerae, Nitrospirae, Planctomycetes, 30 gomonas (and the genus Blastomonas, derived therefrom), Proteobacteria, Spirochaetes, Thermodesulfobacteria, Ther which was created by regrouping organisms belonging to momicrobia, Thermotogae. Thermus (Thermales), or Verru (and previously called species of) the genus Xanthomonas, comicrobia. In an embodiment of a eubacterial host cell, the the genus Acidomonas, which was created by regrouping cell can be a member of any species of eubacteria, excluding organisms belonging to the genus Acetobacter as defined in Cyanobacteria. 35 Bergey (1974). In addition hosts can include cells from the The bacterial host can also be a member of any species of genus Pseudomonas, Pseudomonas enalia (ATCC 14393), Proteobacteria. A proteobacterial host cell can be a member Pseudomonas nigrifaciensi (ATCC 19375), and Pseudomo of any one of the taxa Alphaproteobacteria, Betaproteobac nas putrefaciens (ATCC 8071), which have been reclassified teria, Gammaproteobacteria, Deltaproteobacteria, or Epsi respectively as Alteromonas haloplanktis, Alteromonas nigri lonproteobacteria. In addition, the host can be a member of 40 faciens, and Alteromonas putrefaciens. Similarly, e.g., any one of the taxa Alphaproteobacteria, Betaproteobacteria, Pseudomonas acidovorans (ATCC 15668) and Pseudomonas or Gammaproteobacteria, and a member of any species of testosteroni (ATCC 1 1996) have since been reclassified as Gammaproteobacteria. Comamonas acidovorans and Comamonas testosteroni, In one embodiment of a Gamma Proteobacterial host, the respectively; and Pseudomonas nigrifaciens (ATCC 19375) host will be member of any one of the taxa Aeromonadales, 45 and Pseudomonas piscicida (ATCC 15057) have been reclas Alteromonadales, Enterobacteriales, Pseudomonadales, or sified respectively as Pseudoalteromonas nigrifaciens and Xanthomonadales; or a member of any species of the Entero Pseudoalteromonas piscicida. “Gram-negative Proteobacte bacteriales or Pseudomonadales. In one embodiment, the host ria Subgroup 1 also includes Proteobacteria classified as cell can be of the order Enterobacteriales, the host cell will be belonging to any of the families: Pseudomonadaceae, AZoto a member of the family Enterobacteriaceae, or may be a 50 bacteraceae (now often called by the synonym, the Azoto member of any one of the genera Erwinia, Escherichia, or bacter group' of Pseudomonadaceae), Rhizobiaceae, and Serratia; or a member of the genus Escherichia. Where the Methylomonadaceae (now often called by the synonym, host cell is of the order Pseudomonadales, the host cell may “Methylococcaceae). Consequently, in addition to those be a member of the family Pseudomonadaceae, including the genera otherwise described herein, further Proteobacterial genus Pseudomonas. Gamma Proteobacterial hosts include 55 genera falling within “Gram-negative Proteobacteria Sub members of the species Escherichia coli and members of the group 1 include: 1) Azotobacter group bacteria of the genus species Pseudomonas fluorescens. Azorhizophilus, 2) Pseudomonadaceae family bacteria of the Other Pseudomonas organisms may also be useful. genera Cellvibrio, Oligella, and Teredinibacter; 3) Rhizobi Pseudomonads and closely related species include Gram aceae family bacteria of the genera Chelatobacter, Ensifer, negative Proteobacteria Subgroup 1, which include the group 60 Liberibacter (also called “Candidatus Liberibacter'), and of Proteobacteria belonging to the families and/or genera Sinorhizobium; and 4) Methylococcaceae family bacteria of described as “Gram-Negative Aerobic Rods and Cocci' by R. the genera Methylobacter, Methylocaldum, Methylomicro E. Buchanan and N. E. Gibbons (eds.), Bergey’s Manual of bium, Methylosarcina, and Methylosphaera. Determinative Bacteriology, pp. 217-289 (8th ed., 1974) (The In another embodiment, the host cell is selected from Williams & Wilkins Co., Baltimore, Md., USA) (hereinafter 65 “Gram-negative Proteobacteria Subgroup 2.” “Gram-nega “Bergey (1974)). Table 7 presents these families and genera tive Proteobacteria Subgroup 2 is defined as the group of of organisms. Proteobacteria of the following genera (with the total num US 9,394,571 B2 47 48 bers of catalog-listed, publicly-available, deposited Strains Methylosarcina, Methylosphaera, Azomonas, Azorhizophi thereof indicated in parenthesis, all deposited at ATCC, lus, Azotobacter, Cellvibrio, Oligella, Pseudomonas, Tere except as otherwise indicated): Acidomonas (2); Acetobacter dinibacter, Francisella, Stenotrophomonas, Xanthomonas; (93); Gluconobacter (37); Brevundimonas (23); Beverinckia and Oceanimonas. (13); Derxia (2); Brucella (4); Agrobacterium (79); Chelato In another embodiment, the host cell is selected from bacter (2); Ensifer (3); Rhizobium (144); Sinorhizobium (24); “Gram-negative Proteobacteria Subgroup 5.” “Gram-nega Blastomonas (1): Sphingomonas (27); Alcaligenes (88); Bor tive Proteobacteria Subgroup 5’ is defined as the group of detella (43); Burkholderia (73); Ralstonia (33); Acidovorax Proteobacteria of the following genera: Methylobacter, (20); Hydrogenophaga (9); Zoogloea (9); Methylobacter (2): Methylocaldum, Methylococcus, Methylomicrobium, Methylocaldum (1 at NCIMB); Methylococcus (2); Methylo 10 Methylomonas, Methylosarcina, Methylosphaera, Azomo microbium (2); Methylomonas (9); Methylosarcina (1): nas, Azorhizophilus, Azotobacter, Cellvibrio, Oligella, Methylosphaera, Azomonas (9): Azorhizophilus (5): Azoto Pseudomonas, Teredinibacter, Francisella, Stenotrophomo bacter (64); Cellvibrio (3); Oligella (5); Pseudomonas nas, Xanthomonas; and Oceanimonas. (1139); Francisella (4); Xanthomonas (229); Stenotroph The host cell can be selected from “Gram-negative Proteo Omonas (50); and Oceanimonas (4). 15 bacteria Subgroup 6.” “Gram-negative Proteobacteria Sub Exemplary host cell species of “Gram-negative Proteobac group 6' is defined as the group of Proteobacteria of the teria Subgroup 2 include, but are not limited to the following following genera: Brevundimonas, Blastomonas, Sphin bacteria (with the ATCC or other deposit numbers of exem gomonas, Burkholderia, Ralstonia, Acidovorax, Hydro plary strain(s) thereof shown in parenthesis): Acidomonas genophaga, Azomonas, Azorhizophilus, Azotobacter, methanolica (ATCC 43581); Acetobacter aceti (ATCC Cellvibrio, Oligella, Pseudomonas, Teredinibacter, 15973); Gluconobacter oxydans (ATCC 19357); Brevundi Stenotrophomonas, Xanthomonas; and Oceanimonas. monas diminuta (ATCC 11568); Beijerinckia indica (ATCC The host cell can be selected from “Gram-negative Proteo 9039 and ATCC 19361); Dervia gummosa (ATCC 15994): bacteria Subgroup 7..” “Gram-negative Proteobacteria Sub Brucella melitensis (ATCC 23456), Brucella abortus (ATCC group 7 is defined as the group of Proteobacteria of the 23448); Agrobacterium tumefaciens (ATCC 23308), Agro 25 following genera: Azomonas, Azorhizophilus, Azotobacter, bacterium radiobacter (ATCC 19358), Agrobacterium rhizo Cellvibrio, Oligella, Pseudomonas, Teredinibacter, genes (ATCC 11325); Chelatobacter heintzii (ATCC 29600); Stenotrophomonas, Xanthomonas; and Oceanimonas. Ensifer adhaerens (ATCC 33212); Rhizobium leguminosa The host cell can be selected from “Gram-negative Proteo rum (ATCC 10004); Sinorhizobium fiedi (ATCC 35423); bacteria Subgroup 8.” “Gram-negative Proteobacteria Sub Blastomonas natatoria (ATCC 35951); Sphingomonas 30 group 8' is defined as the group of Proteobacteria of the paucimobilis (ATCC 29837); Alcaligenes faecalis (ATCC following genera: Brevundimonas, Blastomonas, Sphin 8750); Bordetella pertussis (ATCC 9797); Burkholderia gomonas, Burkholderia, Ralstonia, Acidovorax, Hydro cepacia (ATCC 25416); Ralstonia pickettii (ATCC 27511); genophaga, Pseudomonas, Stenotrophomonas, Xanthomo Acidovorax facilis (ATCC 11228); Hydrogenophagafiava nas; and Oceanimonas. (ATCC 33.667); Zoogloea ramigera (ATCC 19544); Methy 35 The host cell can be selected from “Gram-negative Proteo lobacter luteus (ATCC 49878); Methylocaldum gracile bacteria Subgroup 9.” “Gram-negative Proteobacteria Sub (NCIMB 11912); Methylococcus capsulatus (ATCC 19069); group 9” is defined as the group of Proteobacteria of the Methylomicrobium agile (ATCC 35068); Methylomonas following genera: Brevundimonas, Burkholderia, Ralstonia, methanica (ATCC 35067); Methylosarcina fibrata (ATCC Acidovorax, Hydrogenophaga, Pseudomonas, Stenotroph 700909); Methylosphaera hansonii (ACAM 549): Azomonas 40 Omonas; and Oceanimonas. agilis (ATCC 7494): Azorhizophilus paspali (ATCC 23833); The host cell can be selected from “Gram-negative Proteo Azotobacter chroococcum (ATCC 9043); Cellvibrio mixtus bacteria Subgroup 10.” “Gram-negative Proteobacteria Sub (UQM2601); Oligella urethralis (ATCC 17960); Pseudomo group 10' is defined as the group of Proteobacteria of the nas aeruginosa (ATCC 10145), Pseudomonas fluorescens following genera: Burkholderia, Ralstonia, Pseudomonas, (ATCC 35858): Francisella tularensis (ATCC 6223): 45 Stenotrophomonas; and Xanthomonas. Stenotrophomonas maltophilia (ATCC 13637); Xanthomo The host cell can be selected from “Gram-negative Proteo nas campestris (ATCC 33913); and Oceanimonas doudorofii bacteria Subgroup 11.” “Gram-negative Proteobacteria Sub (ATCC 27123). group 11 is defined as the group of Proteobacteria of the In another embodiment, the host cell is selected from genera: Pseudomonas, Stenotrophomonas; and Xanthomo “Gram-negative Proteobacteria Subgroup 3.” “Gram-nega 50 nas. The host cell can be selected from “Gram-negative Pro tive Proteobacteria Subgroup 3 is defined as the group of teobacteria Subgroup 12.” “Gram-negative Proteobacteria Proteobacteria of the following genera: Brevundimonas, Subgroup 12 is defined as the group of Proteobacteria of the Agrobacterium, Rhizobium, Sinorhizobium, Blastomonas, following genera: Burkholderia, Ralstonia, Pseudomonas. Sphingomonas, Alcaligenes, Burkholderia, Ralstonia, Aci The host cell can be selected from “Gram-negative Proteo dovorax, Hydrogenophaga, Methylobacter, Methylocal 55 bacteria Subgroup 13.” “Gram-negative Proteobacteria Sub dum, Methylococcus, Methylomicrobium, Methylomonas, group 13 is defined as the group of Proteobacteria of the Methylosarcina, Methylosphaera, Azomonas, Azorhizophi following genera: Burkholderia, Ralstonia, Pseudomonas; lus, Azotobacter, Cellvibrio, Oligella, Pseudomonas, Tere and Xanthomonas. The host cell can be selected from "Gram dinibacter, Francisella, Stenotrophomonas, Xanthomonas; negative Proteobacteria Subgroup 14.” “Gram-negative Pro and Oceanimonas. 60 teobacteria Subgroup 14' is defined as the group of Proteo In another embodiment, the host cell is selected from bacteria of the following genera: Pseudomonas and “Gram-negative Proteobacteria Subgroup 4.” “Gram-nega Xanthomonas. The host cell can be selected from "Gram tive Proteobacteria Subgroup 4” is defined as the group of negative Proteobacteria Subgroup 15.” “Gram-negative Pro Proteobacteria of the following genera: Brevundimonas, teobacteria Subgroup 15” is defined as the group of Proteo Blastomonas, Sphingomonas, Burkholderia, Ralstonia, Aci 65 bacteria of the genus Pseudomonas. dovorax, Hydrogenophaga, Methylobacter, Methylocal The host cell can be selected from “Gram-negative Proteo dum, Methylococcus, Methylomicrobium, Methylomonas, bacteria Subgroup 16.” “Gram-negative Proteobacteria Sub US 9,394,571 B2 49 50 group 16' is defined as the group of Proteobacteria of the The host cell can be selected from “Gram-negative Proteo following Pseudomonas species (with the ATCC or other bacteria Subgroup 17.” “Gram-negative Proteobacteria Sub deposit numbers of exemplary strain(s) shown in parenthe group 17 is defined as the group of Proteobacteria known in sis): Pseudomonas abietaniphila (ATCC 700689); the art as the “fluorescent Pseudomonads' including those Pseudomonas aeruginosa (ATCC 101.45); Pseudomonas belonging, e.g., to the following Pseudomonas species: alcaligenes (ATCC 14909); Pseudomonas anguilliseptica 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, 10 Pseudomonas mandelii, Pseudomonas marginalis, 33634); Pseudomonas oleovorans (ATCC 8062); Pseudomo Pseudomonas migulae, Pseudomonas mucidolens, nas pseudoalcaligenes (ATCC 17440); Pseudomonas resino Pseudomonas Orientalis, Pseudomonas rhodesiae, vorans (ATCC 14235); Pseudomonas straminea (ATCC Pseudomonas synxantha, Pseudomonas tolaasii; and 33.636); Pseudomonas agarici (ATCC 25941); Pseudomonas Pseudomonas veronii. alcaliphila, Pseudomonas alginovora, Pseudomonas ander 15 Other suitable hosts include those classified in other parts sonii. Pseudomonas aspleni (ATCC 23835); Pseudomonas of the reference, such as Gram (+) Proteobacteria. In one azelaica (ATCC 27162): Pseudomonas beverinckii (ATCC embodiment, the host cell is an E. coli. The genome sequence 19372); Pseudomonas borealis, Pseudomonas boreopolis for E. coli has been established for E. coli MG1655 (Blattner, (ATCC 33662); Pseudomonas brassicacearum, Pseudomo et al. (1997) The complete genome sequence of Escherichia nas butanovora (ATCC 43655); Pseudomonas cellulosa coli K-12, Science 277(5331): 1453-74) and DNA microar (ATCC 55703); Pseudomonas aurantiaca (ATCC 33663); rays are available commercially for E. coli K12 (MWG Inc. Pseudomonas chlororaphis (ATCC 9446, ATCC 13985, High Point, N.C.). E. coli can be cultured in either a rich ATCC 17418, ATCC 17461); Pseudomonas fragi (ATCC medium such as Luria-Bertani (LB) (10 g/L tryptone, 5 g/L 4973); Pseudomonas lundensis (ATCC 49968); Pseudomo NaCl, 5 g/L yeast extract) or a defined minimal medium such nas taetrolens (ATCC 4683); Pseudomonascissicola (ATCC 25 as M9 (6g/L NaHPO3 g/L KHPO 1 g/L NHCl, 0.5g/L 33616); Pseudomonas coronafaciens, Pseudomonas diter NaCl, pH 7.4) with an appropriate carbon source such as 1% peniphila, Pseudomonas elongata (ATCC 10144); glucose. Routinely, an over night culture of E. coli cells is Pseudomonas flectens (ATCC 12775); Pseudomonas azoto diluted and inoculated into fresh rich or minimal medium in formans, Pseudomonas brenneri. Pseudomonas cedrella, either a shake flask or a fermentor and grown at 37° C. Pseudomonas corrugata (ATCC 29736); Pseudomonas 30 A host cell can also be of mammalian origin, Such as a cell extremorientalis, Pseudomonas fluorescens (ATCC 35858); derived from a mammal including any human or non-human Pseudomonas gessardii. Pseudomonas libanensis, mammal. Mammals can include, but are not limited to pri Pseudomonas mandelii (ATCC 700871); Pseudomonas mar mates, monkeys, porcine, ovine, bovine, rodents, ungulates, ginalis (ATCC 10844); Pseudomonas migulae, Pseudomo pigs, Swine, sheep, lambs, goats, cattle, deer, mules, horses, nas mucidolens (ATCC 4685); Pseudomonas orientalis, 35 monkeys, apes, dogs, cats, rats, and mice. Pseudomonas rhodesiae, Pseudomonas synxantha (ATCC A host cell may also be of plant origin. Cells from any plant 9890); Pseudomonas tolaasii (ATCC 33618); Pseudomonas can be selected in which to screen for the production of a veronii (ATCC 700474); Pseudomonas federiksbergensis, heterologous protein of interest. Examples of Suitable plant Pseudomonas geniculata (ATCC 19374); Pseudomonas gin include, but are not limited to, alfalfa, apple, apricot, Arabi geri. Pseudomonas graminis, Pseudomonas grimontii, 40 dopsis, artichoke, arugula, asparagus, avocado, banana, bar Pseudomonas halodenitrificans, Pseudomonas halophila, ley, beans, beet, blackberry, blueberry, broccoli, brussels Pseudomonas hibiscicola (ATCC 19867); Pseudomonas hut Sprouts, cabbage, canola, cantaloupe, carrot, cassaya, castor tiensis (ATCC 14670); Pseudomonas hydrogenovora, bean, cauliflower, celery, cherry, chicory, cilantro, citrus, Pseudomonas jessenii (ATCC 700870); Pseudomonas kilon clementines, clover, coconut, coffee, corn, cotton, cranberry, ensis, Pseudomonas lanceolata (ATCC 14669); Pseudomo 45 cucumber, Douglas fir, eggplant, endive, escarole, eucalyp nas lini. Pseudomonas marginata (ATCC 25417); tus, fennel, figs, garlic, gourd, grape, grapefruit, honey dew, Pseudomonas mephitica (ATCC 33665); Pseudomonas deni jicama, kiwifruit, lettuce, leeks, lemon, lime, Loblolly pine, trificans (ATCC 19244); Pseudomonas pertucinogena linseed, mango, melon, mushroom, nectarine, nut, oat, oil (ATCC 190); Pseudomonas pictorum (ATCC 23328); palm, oil seed rape, okra, olive, onion, orange, an ornamental Pseudomonas psychrophila, Pseudomonas filva (ATCC 50 plant, palm, papaya, parsley, parsnip, pea, peach, peanut, 31418); Pseudomonas monteilii (ATCC 700476); Pseudomo pear, pepper, persimmon, pine, pineapple, plantain, plum, nas mosselii, Pseudomonas oryzihabitans (ATCC 43272): pomegranate, poplar, potato, pumpkin, quince, radiata pine, Pseudomonas plecoglossicida (ATCC 700383); Pseudomo radiscChio, radish, rapeseed, raspberry, rice, rye, Sorghum, nas putida (ATCC 12633); Pseudomonas reactans, Southern pine, soybean, spinach, Squash, Strawberry, Sugar Pseudomonas spinosa (ATCC 14606); Pseudomonas bale 55 beet, Sugarcane, Sunflower, Sweet potato, Sweetgum, tanger arica, Pseudomonas luteola (ATCC 43273); Pseudomonas ine, tea, tobacco, tomato, triticale, turf, turnip, a vine, water Stutzeri (ATCC 17588); Pseudomonas amygdali (ATCC melon, wheat, yams, and Zucchini. In some embodiments, 33614); Pseudomonas avellanae (ATCC 700331); plants useful in the method are Arabidopsis, corn, wheat, Pseudomonas caricapapayae (ATCC 33615); Pseudomonas Soybean, and cotton. cichorii (ATCC 10857); Pseudomonas ficuserectae (ATCC 60 Kits 35104); Pseudomonas fiscovaginae, Pseudomonas meliae The present invention also provides kits useful for identi (ATCC 33050); Pseudomonas syringae (ATCC 19310): fying a host strain, e.g. a Pfluorescens host strain, optimal for Pseudomonas viridiflava (ATCC 13223); Pseudomonas ther producing a heterologous protein or polypeptide of interest. mocarboxydovorans (ATCC 35961); Pseudomonas thermo The kit comprises a plurality of phenotypically distinct host tolerans, Pseudomonas thivervalensis, Pseudomonas van 65 cells, wherein each population has been genetically modified couverensis (ATCC 700688); Pseudomonas wisconsinensis; to increase the expression of one or more target genes and Pseudomonas Xiamenensis. involved in protein production, to decrease the expression of US 9,394,571 B2 51 52 one or more target genes involved in protein degradation, or transformation of plasmids that express the heterologous pro both. The array may further comprise one or more popula tein of interest, were screened for improved protein yield tions of cells that have not been genetically modified to modu and/or quality. late the expression of either a gene involved in protein pro duction or a gene involved in protein degradation. These kits Example 1 may also comprise reagents sufficient to facilitate growth and maintenance of the cell populations as well as reagents and/or Identification of Folding Modulator Genes in the constructs for expression of a heterologous protein or Genome of Pfluorescens Strain MB214 polypeptide of interest. The populations of host cells may be provided in the kit in any manner Suitable for storage, trans 10 Folding modulators are a class of proteins present in all port, and reconstitution of cell populations. The cell popula cells which aid in the folding, unfolding and degradation of tions may be provided live in a tube, on a plate, or on a Slant, nascent and heterologous polypeptides. Folding modulators or may be preserved either freeze-dried or frozen in a tube or include chaperones, chaperoning, peptidyl-prolyl cis-trans vial. The cell populations may contain additional components 15 isomerases, and proteins involved in protein disulfide bond in the storage media Such as glycerol. Sucrose, albumin, or formation. As a first step to construct novel production strains other Suitable protective or storage agents. with the ability to help fold heterologous proteins, the P. The following examples are offered by way of illustration fluorescens genome was mined to identify host cell folding and not by way of limitation. modulator genes. Each of the 6,433 predicted ORFs of the Pfluorescens EXPERIMENTAL, EXAMPLES MB214 genome was analyzed for the possibility that they encoded a folding modulator using the following method. Overview Several folding modulators of interest had already been iden Heterologous protein production often leads to the forma tified by Dow researchers by analysis of the genome annota tion of insoluble or improperly folded proteins, which are 25 tion (Ramseier et. al. 2001). Homologs of these starting pro difficult to recover and may be inactive. Furthermore, the teins were identified using protein/protein BLAST with the presence of specific host cell proteases may degrade the pro starting protein as the query and a database of all MB214 tein of interest and thus reduce the final yield. There is no translated ORFs as the subject. Those translated ORFs which single factor that will improve the production of all heterolo matched the query proteins with significant homology were gous proteins. Thus, a method was sought to identify factors 30 added to the list for further analysis. Significant homology is specific to a particular heterologous protein from a pool of defined here as having an e-score of 1e-30 or less with allow likely candidates. ances made for human judgment based on the length and Using Systems Biology tools, the Pfluorescens genome quality of the alignment. The intention of this study was to be was mined to identify host cell protein folding modulator and very inclusive to maximize the chance that all potential fold protease genes. Then, global gene expression analyses were 35 ing modulators would be identified. performed to prioritize upregulated targets, and, thereafter, More ORFs were added to the list based on their curated novel protein production strains were constructed. As a result, function from the previous annotation containing the key a “Pfenex Strain Array' was assembled consisting of a plu word “chaperone'. Finally, the ORFs were analyzed by the rality of phenotypically distinct Pfluorescens hoststrains that 40 protein signature family searching program InterProScan are deficient in host-cell proteases or allow the co-overex (Quevillon et. al. 2005) against the InterPro Database version pression of protein folding modulators. This strain array can 7.0 (Mulder et. al. 2005). The ORFs were assigned protein be used to screen for factors that specifically enhance the families by the InterProScan software as well as Gene Ontol yield or quality of certain heterologous proteins. Providing a ogy (GO) categories associated with those families (Gene plurality of phenotypically distinct host strains increases the 45 Ontology Consortium. 2004). Using these automatic GO chance of success of identifying a hoststrain that will increase assignments, all of the ORFs which had been assigned the GO the production of any individual heterologous protein of terms “GO:0006457 Biological Process: protein folding or interest. “GO:0003754 Molecular Function: chaperone activity” were This invention provides an improvement in the production added to the list for further analysis. of heterologous proteins in Pseudomonas fluorescens. Hav 50 The list was then analyzed to remove ORFs which had a ing available a library of host strains in the same genetic low probability of encoding folding modulators. Again, the background allows the rapid screening and identification of intent of this study was to be very inclusive but many of the factors that increase the yield and/or quality of heterolo ORFs assigned to the list by these semi-automated methods gously expressed proteins. The genome sequence of Pfluo could be easily identified as not coding for folding modula rescens has been annotated and targeted host cell folding 55 tors based on limited criteria and human judgment. modulators and proteases have been identified. Folding The most common reason for excluding a certain ORF was modulators assist in the proper folding of proteins and include the weak evidence that this ORF is actually a folding modu chaperones, chaperoning, peptidyl-proline isomerases (PPI lator, i.e. ORFs which had been assigned to the list based on ases), and disulfide bond formation proteins. Proteases can the previous annotation where the reasoning for annotating degrade the protein of interest and thus affect heterologous 60 the ORF as a folding modulator was either unclear or contra protein yield and quality. Using background knowledge from dictory. InterProScan is actually a conglomerate of different the literature and DNA microarray analyses to identify likely programs and some of these programs are considered to be targets, a list of about 80 target genes was assembled. In host more reliable than others. If an ORF was assigned to the list cells that have the same genetic background, these genes were based solely on the output of the ScanRegExp or ProfileScan either removed from the genome or cloned into plasmids to 65 components then it was removed. The final list of Pfluore enable co-overexpression along with heterologous proteins. scens folding modulators has 43 members and is shown in The resulting strains were arrayed in 96-well format and, after Table 1. US 9,394,571 B2 53 54 Example 2 protease homolog but not a protease itself. The final list of P fluorescens proteases has 90 members and is shown in Table Identification of Protease Genes in the Genome of P 2. fluorescens Strain MB214 Example 3 Proteases are enzymes that hydrolyze peptide bonds and are necessary for the survival of all living creatures. However, In Silico Cellular Location Prediction of the Folding their role in the cell means that proteases can be detrimental to Modulator and Protease Proteins recombinant protein yield and/or quality in any heterologous protein expression system, which also includes the PfeneX 10 One of the strengths of the Pfenex Expression Technol Expression TechnologyTM. As a first step to construct novel ogyTM is its ability to control the cellular compartment to production strains that have protease genes removed from the which a particular heterologous protein can be segregated. genome, the Pfluorescens genome was mined to identify host Thus, the cellular compartments where the identified host cell cell protease genes. folding modulator and protease proteins are located were 15 predicted. To make these predictions, two programs were Each of the 6,433 predicted ORFs of the Pfluorescens chosen. PsortB 2.0 combines the results of 12 separate algo MB214 genome were analyzed for the possibility that they rithms, which predict the Subcellular location of a given pep encoded a protease using the following method. The tide. The majority of the algorithms rely on detecting homol MEROPS database is manually curated by researchers at the ogy between the query protein and proteins of known Wellcome Trust Sanger Institute, Cambridge, UK (Rawlings subcellular localization. PsortBalso includes algorithms such et. al., 2006, Nucleic Acids Research 34 (Database issue): as HMMTOP and SignalP, which detect the presence of trans D270-2). It is a comprehensive list of proteases discovered membrane folding domains or type I Secretion signal both through laboratory experiments as well as by homology sequences, respectively, using Hidden Markov Models to known protease families. One of the strengths of the data (HMM). In addition to the PsortB results, SignalPHMM was base is the MEROPS hierarchical classification scheme. In 25 used to predict the presence of type I Secretion signal this system, homologs which share the same function are sequences. This was necessary because the output of PsortB grouped together into families. Families are grouped into can be vague when a signal sequence is detected but no other clans based on evolutionary relatedness that again are based specific information indicating the Subcellular location is on similar structural characteristics. The method makes great given. In these cases, PsortB indicates that the subcellular use of the database to identify protease homologs within the 30 localization of the protein is unknown, because it really could Pfluorescens genome. segregate to any one of the cytoplasmic membrane, peri Homologs to the MEROPS database were identified using plasm, outer membrane or extracellular compartments. How protein/protein BLAST with each MB214 translated ORF as ever, it is informative enough to know that the protein is the query and a database of all of the MEROPS proteins as the probably not located in the cytoplasm to make it worth noting subject. Those translated ORFs, which matched the query 35 that in the table. Thus, Table 2 lists the results of the PsortB proteins with significant homology, were added to the list for algorithm except in cases where that result was unknown. In further analysis. Significant homology in this case is defined these cases the result of SignalP HMM alone is given with here as having an e-score of 1e" or less with allowances “Signal Peptide' indicating that a signal peptide was detected made for human judgment based on the length and quality of and "Non Secretory indicating that no signal peptide was the alignment. This step yielded 109 potential proteases for 40 detected. the list. The ORFs were also analyzed by the protein signature Example 4 family searching program InterProScan (Quevillon et. al. 2005) against the InterPro Database version 7.0 (Mulder et. Construction of Plasmids that Enable the al. 2005). The ORFs were assigned protein families by the 45 Co-Overexpression of Folding Modulators InterProScan software as well as Gene Ontology (GO) cat egories associated with those families (Gene Ontology Con Folding modulator genes were cloned into a plasmid sortium. 2004). Using these automatic GOassignments, all of derivative of pCN (Nieto et al. 1990), which is compatible the ORFs which had been assigned a GO name that contained with another plasmid that routinely is used to express the the strings "peptidase”, “protease' or “proteolysis” were 50 heterologous protein of interest (Squires et al. 2004; Chew et added to the list for further analysis. This step yielded an al. 2005). The construction of a mannitol-inducible grpE additional 70 potential proteases that had not been identified dnaKJ-containing plasmid is exemplified. Other folding in the previous step. modulators—either as a single gene or as multiple genes More ORFs were added to the list based on their curated when organized in operons—were cloned similarly as out function from the previous annotation (Ramseier et. al. 2001) 55 lined below. containing the keywords "peptidase' or “protease'. This step Employing genomic DNA isolated from P. fluorescens yielded 32 potential proteases that again had not been iden MB214 (DNeasy; Qiagen, Valencia, Calif.) as a template and tified in the previous steps. primers RC199 The list was then analyzed to remove ORFs which had a (5-ATATACTAGTAGGAGGTAACTTATGGCT low probability of encoding proteases. Again, the intent of 60 GACGAACAGACGCA-3) (SEQID NO:1) and RC200 this study was to be very inclusive but many of the ORFs (5'-ATATTCTAGATTACAGGTCGCCGAAGAAGC-3) assigned to the list by these semi-automated methods could (SEQID NO:2), the grpE-dnaKJ genes were amplified using be easily identified as not coding for proteases based on PfuTurbo (Stratagene, La Jolla, Calif.) as per the manufac limited criteria and human judgment. The two most common turer's recommendations. The resulting 4 kb PCR product reasons for excluding genes were the weak evidence that a 65 was digested with Spel and Xbal (restriction sites underlined certain ORF is actually a protease, or that a particular gene in the primers above) and ligated into pl)OW2236 which is a showed greatest homology with another protein known to be derivative of pDOW 1306-6 (Schneideretal. 2005b) to create US 9,394,571 B2 55 56 pDOW2240 containing the grpE-dnaKJ operon under control Microbial and Eucarvotic Expression Systems. G. Gellis of the tac promoter. Plasmid pl)OW2240 was then digested sen. Weinheim, WILEY-VCH: 45-66 with Speland HindIII and the resulting grpE-dnaKJ-contain Dolinski, K, Heitman, J. 1997. Peptidyl-prolyl isomerases— ing 4.0 kb DNA fragment was gel-purified using Qiaquick an overview of the cyclophilin, FKBP and parvulin fami (Qiagen, Valencia, Calif.) and ligated into pl)OW2247, lies. in Guidebook to Molecular Chaperones and Protein which is a derivative of pCN carrying the Pfluorescens man Folding Catalysts. Gething M-J. Ed. Oxford University nitol-regulated promoter (Schneider et al. 2005a), that was Press Inc., New York: 359-369 also digested with Spel and HindIII. The resulting plasmid, Gardy, J. L., M. R. Laird, F. Chen, S. Rey, C. J. Walsh, M. pDOW3501, contained the grpE-dnaKJ operon under the Ester, and F. S. L. Brinkman 2005 PSORTb v.2.0: control of the mannitol promoter. Plasmid plCW3501 was 10 expanded prediction of bacterial protein subcellular local then transformed into DC388 and other uracil-auxotrophic ization and insights gained from comparative proteome strains by selecting on M9 glucose plates Supplemented with analysis. Bioinformatics 21(5):617-623. 250 ug/ml uracil. Gene Ontology Consortium. 2004. The Gene Ontology (GO) 15 database and informatics resource. Nucleic Acids Example 5 Research 32: D258-D261. Gething M-J. Ed. 1997. Guidebook to Molecular Chaperones Construction of Pfluorescens Strains with Genomic and Protein-Folding Catalysts. Oxford University Press Deletions of Protease Genes Inc., New York. Horton, R. M., Z. Cai, S.N. Ho and L. R. Pease (1990). “Gene Plasmids that enabled the creation of genomic deletions splicing by overlap extension: tailor-made genes using the were constructed by amplification of 500-1000 bp DNA frag polymerase chain reaction.” BioTechniques 8(5): 528-30, ments both 5' and 3' of the gene to be deleted. The resulting 5' 532,534-5 PCR product typically ends with the translational initiation Lombardo, M-J, Thanassi, DG, Hultgren, S.J. 1997. Escheri codon (ATG or GTG or TGT) of the gene to be deleted while 25 chia coli PapD. in Guidebook to Molecular Chaperones the 3'PCR product typically begins with the stop codon (TAA and Protein-Folding Catalysts. Gething M-J. Ed. Oxford or TGA or TAG) of the gene to be deleted. These two PCR University Press Inc., New York: 463-465 products were fused together through an additional amplifi Mulder NJ. Apweiler R, Attwood T K. Bairoch A, Bateman cation step then cloned into pI)OW1261 (FIG.1) (Chew etal. A, Binns D, Bradley P. Bork P. Bucher P. Cerutti L, Copley 2005) using SOE PCR (Horton et al. 1990). 30 R. Courcelle E. Das U. Durbin R, Fleischmann W. Gough J. Haft D. Harte N. Hulo N. Kahn D. Kanapin A, Example 6 Krestyaminova M, Lonsdale D. Lopez R. Letunic I, Mad era M, Maslen J. McDowall J. Mitchell A, Nikolskaya AN, High-Through-Put Growth and Analysis of Orchard S. Pagni M. Ponting CP, Quevillon E. Selengut J. Heterologous Protein Expression in Pfluorescens 35 Sigrist CJ, Silventoinen V. Studholme DJ. Vaughan R. Wu Strains C H. 2005. InterPro, Progress and Status in 2005. Nucleic Acids Res. 33, Database Issue:D201-5. Plasmid pl)OW2787 encodes the monoclonal antibody Nieto, C. E. Femandez-Tresguerres, N. Sanchez, M. Vicente (m-Ab) gal2; the heavy chain is expressed with a Pbp secre and R. Diaz (1990). “Cloning vectors, derived from a natu tion leader and under control of the tac promoter. The light 40 rally occurring plasmid of Pseudomonas savastanoi, spe chain is expressed with an OprF secretion leader and under cifically tailored for genetic manipulations in Pseudomo control of the mannitol promoter. The plasmid was electropo nas.' Gene 87(1): 145-9. rated into competent cells of 63 strains carrying either a Quevillon E., Silventoinen V., Pillai S., Harte N., Mulder N., directed gene deletion or pl)OW2247 carrying a folding Apweiler R. Lopez R. (2005) InterProScan: protein modulator for co-expression, and five control strains contain 45 domains identifier. Nucleic Acids Research 33: W116 ing a wild type strain. Cells were cultured in replicate deep W12O. well blocks containing growth medium with glycerol by Ramseier T M. S. C., Payne J, Chew L. Rothman L. D. Sub shaking at 300 rpm. Protein expression was induced at 24 hrs ramanian M. 2001. The Pseudomonas fluorescens MB214 with 0.1 mM isopropyl B-D-thiogalactopyranoside (IPTG) Genome Sequence. CRI CRI2001001442: BIOTECH and 1% mannitol. At 24 hrs post-induction, aliquots were 50 01-007. The Dow Chemical Company. lysed, antigen-binding of the antigen was measured to quan Ranson, N. A. White, H E. Saibil, H R. 1998. Chaperonins titate amounts of active antibody. The value was divided by Biochem. J.333, 233-242. ODoo to measure cell specific activity. Strains Aprc1. Rawlings, N. D., Morton, F. R. & Barrett, A. J. 2006. AdegP2, ALa2, AclpP, and Aprc2, Aprc2, the grpEdnaKJ co MEROPS: the peptidase database. Nucleic Acids Res 34, expression Strain, Atig, AclpX, and Alon were all 2.4-fold or 55 D270-D272. more higher than the control strains, which was statistically Schneider, J. C., A. F. Jenings, D. M. Mun, P. M. McGovern significant (p<0.5). Soluble cells fractions were prepared and L. C. Chew (2005a). “Auxotrophic markers pyrF and from Aprc.1, AdegP2, ALa2 and the grpEdnaKJ co-expression proC can replace antibiotic markers on protein production strain and subjected to Western analysis (FIG.2). A band with plasmids in high-cell-density Pseudomonas fluorescens a size consistent with fully assembled antibody was detected 60 fermentation.” Biotechnology Progress 21(2): 343-348. in the four test strains, but not in the control. Schneider, J. C., B. 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