- 1 AUSTRALIA PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT ORIGINAL Name of Applicant: Pfenex, Inc. Actual Inventors: Diane M. Retallack and Lawrence Chew and Hongfan Jin Address for Service is: SHELSTON IP 60 Margaret Street Telephone No: (02) 9777 1111 SYDNEY NSW 2000 Facsimile No. (02) 9241 4666 CCN: 3710000352 Attorney Code: SW Invention Title: HIGH LEVEL EXPRESSION OF RECOMBINANT CRM197 The following statement is a full description of this invention, including the best method of performing it known to me/us: File: 65814AUP00 HIGH LEVEL EXPRESSION OF RECOMBINANT CRM197 BACKGROUND OF THE INVENTION [000a] The present application claims priority from U. S. Provisional Patent Application Ser. No. 61/319,152, filed March 30, 2010, incorporated in its entirety herein by reference. 5 [000b] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field. 100011 Diphtheria toxin (DT) is a proteinaceous toxin that is synthesized and secreted by toxigenic strains of Corynebacterium diphtheriae. Toxigenic strains contain a bacteriophage lysogen 10 carrying the toxin gene. DT is synthesized as a 535-amino-acid polypeptide, which undergoes proteolysis to form the mature toxin. The mature toxin comprises two subunits, A and B,joined by a disulfide bridge. The B subunit, formed from the C-terminal portion of intact DT, enables binding and entry of DT through the cell membrane and into the cytoplasm. Upon cell entry, the enzymatic A subunit, formed from the N terminal portion of 15 intact DT, catalyzes ADP ribosylation of Elongation Factor 2 (EF-2). As a result, EF-2 is inactivated, protein synthesis stops, and the cell dies. Diphtheria toxin is highly cytotoxic; a single molecule can be lethal to a cell, and a dose of 10 ng/kg can kill animals and humans. [00021 The CRM 197 protein is a nontoxic, immunologically cross-reacting form of DT. It has been studied for its potential use as a DT booster or vaccine antigen. CRM 197 is produced by C. 20 diphtheriae that has been infected by the nontoxigenic phage p197'0 created by nitrosoguanidine mutagenesis of the toxigenic corynephage P. The CRM197 protein has the same molecular weight as DT but differs by a single base change (guanine to adenine) in the A subunit. This single base change results in an amino acid substitution (glutamic acid for glycine) and eliminates the toxic properties of DT. 25 100031 Conjugated polysaccharide vaccines that use CRM 197 as a carrier protein have been approved for human use. Vaccines include: Menveo* (Novartis Vaccines and Diagnostics), a vaccine indicated for preventing invasive meningococcal disease caused by Neisseria meningitidis subgroups A, C, Y, and W-l35; Menjugate (Novartis Vaccines), a meningococcal group C conjugate vaccine; and Prevnar* (Wyeth Pharmaceuticals, Inc.), a 30 childhood pneumonia vaccine that targets seven serotypes of Streptococcus pneumoniae, and HibTITER*(Wyeth), a Haemophilus influenzae type b vaccine. In addition, CRM 197 2 has potential use as a boosting antigen for diphtheria and is being investigated as a carrier protein for use in other vaccines. 100041 A method for high-level expression of CRM 197 for approved therapeutics and investigational use has not been reported. CRM 197 has been expressed in, e.g., C. 5 diphtheriae, B. subtilis, and E. coli, at levels that range in the tens of mg/L. A single dose of the Prevnar conjugate vaccine contains about 20 tg of CRM197. Therefore, a method for economically producing CRM 197 at levels of about I g/L or more would greatly facilitate vaccine research and manufacture. SUMMARY OF THE INVENTION 10 100051 According to a first aspect of the invention there is provided a method for producing a recombinant CRM 197 protein in a Pseudomonas host cell, said method comprising: ligating into an expression vector a nucleotide sequence encoding a CRM 197 protein fused to a secretion signal that directs transfer of the CRM 197 protein to the periplasm; I 5 transforming the Pseudomonas host cell with the expression vector; and culturing the transformed Pseudomonas host cell in a culture media suitable for the expression of the recombinant CRM 197 protein; wherein the yield of soluble CRM 197 obtained is about 0.5 grams per liter to about 12 grams per liter. 20 100061 In embodiments, the Pseudomonas host cell is defective in the expression of at least one protease or the Pseudomonas host cell overexpresses at least one folding modulator. In certain embodiments, the Pseudomonas host cell is hslUV-, prcl-, degPl-, degP2-, and aprA-. In embodiments, the the Pseudomonas host cell is hsIUV-,prcI-, degPl-, degP2-, and aprA-, and the secretion leader is Azu, lbpS3 IA, CupA2, or PbpA20V. In other 25 embodiments, the Pseudomonas host cell is hsIUV-, prcl-, degPl-, degP2-, and aprA-, and the secretion leader is Azu, IbpS3 IA, CupA2, PbpA20V, or Pbp. In other embodiments, the Pseudomonas host cell is defective in the expression of Serralysin, HsIU, HsIV, Prcl, DegP I, DegP2, or AprA, or the Pseudomonas host cell overexpresses DsbA, DsbB, DsbC, and DsbD. 30 10007] In specific embodiments, the host cell overexpresses DsbA, DsbB, DsbC, and DsbD, and the secretion leader is Azu. In other specific embodiments, the host cell is defective in the expression of Serralysin, and the secretion leader is Pbp or Azu. In certain embodiments the 3 host cell is defective in the expression of HsIU and HsIV, and the secretion leader is Pbp or Azu. In still other embodiments, the Pseudomonas host cell is wild-type and the secretion leader is Pbp or Azu. 10008] In embodiments, the secretion leader is Azu, Pbp, lbpS3 I A, CupA2, or PbpA20V. In other 5 embodiments, the secretion leader is Azu, IbpS3 IA, CupA2, or PbpA20V. 100091 In embodiments, the CRM 197 nucleotide sequence has been optimized for expression in the Pseudomonas host cell. 100101 In embodiments, the yield of soluble CRM 197 obtained is about 0.5 g/L, about 0.6 g/L, about 0.7 g/L, about 0.8 g/L, about 0.9 g/L, about I g/L, about 1.5 g/L, about 2 g/L, about 10 2.5 g/L, about 3 g/L, about 3.5 g/L, about 4 g/L, about 4.5 g/L, about 5 g/L, about 5.5 g/L, about 6 g/L, about 6.5 g/L, about 7 g/L, about 7.5 g/L, about 8 g/L, about 8.5 g/L, about 9 g/L, about 9.5 g/L, about 10 g/L, about 10.5 g/L, about I I g/L, about 12 g/L, about 0.5 g/L to about I g/L, about 0.5 g/L to about 2 g/L, about 0.5 g/L to about 3 g/L, about 0.5 g/L to about 4 g/L, about 0.5 g/L to about 5 g/L, about 0.5 g/L to about 6 g/L, about 0.5 g/L to 15 about 7 g/L, about 0.5 g/L to about 8 g/L, about 0.5 g/L to about 9 g/L, about 0.5 g/L to about 10 g/L, about 0.5 g/L to about I I g/L, about 0.5 g/L to about 12 g/L, about I g/L to about 2 g/L, about I g/L to about 3 g/L, about I g/L to about 4 g/L, about I g/L to about 5 g/L, about I g/L to about 6 g/L, about I g/L to about 7 g/L, about I g/L to about 8 g/L, about 1 g/L to about 9 g/L, about I g/L to about 10 g/L, about I g/L to about I g/L, about I g/L 20 to about 12 g/L, about 2 g/L to about 3 g/L, about 2 g/L to about 4 g/L, about 2 g/L to about 5 g/L, about 2 g/L to about 6 g/L, about 2 g/L to about 7 g/L, about 2 g/L to about 8 g/L, about 2 g/L to about 9 g/L, about 2 g/L to about 10 g/L, about 2 g/L to about I I g/L, about 2 g/L to about 12 g/L, about 3 g/L to about 4 g/L, about 3 g/L to about 5 g/L, about 3 g/L to about 6 g/L, about 3 g/L to about 7 g/L, about 3 g/L to about 8 g/L, about 3 g/L to about 9 25 g/L, about 3 g/L to about 10 g/L, about 3 g/L to about I I g/L, about 3 g/L to about 12 g/L, about 4 g/L to about 5 g/L, about 4 g/L to about 6 g/L, about 4 g/L to about 7 g/L, about 4 g/L to about 8 g/L, about 4 g/L to about 9 g/L, about 4 g/L to about 10 g/L, about 4 g/L to about 11 g/L, about 4 g/L to about 12 g/L, about 5 g/L to about 6 g/L, about 5 g/L to about 7 g/L, about 5 g/L to about 8 g/L, about 5 g/L to about 9 g/L, about 5 g/L to about 10 g/L, 30 about 5 g/L to about I I g/L, about 5 g/L to about 12 g/L, about 6 g/L to about 7 g/L, about 6 g/L to about 8 g/L, about 6 g/L to about 9 g/L, about 6 g/L to about 10 g/L, about 6 g/L to about I I g/L, about 6 g/L to about 12 g/L, about 7 g/L to about 8 g/L, about 7 g/L to about 9 g/L, about 7 g/L to about 10 g/L, about 7 g/L to about 1I g/L, about 7 g/L to about 12 g/L, 4 about 8 g/L to about 9 g/L, about 8 g/L to about 10 g/L, about 8 g/L to about I I g/L, about 8 g/L to about 12 g/L, about 9 g/L to about 10 g/L, about 9 g/L to about I I g/L, about 9 g/L to about 12 g/L, about 10 g/L to about I I g/L, about 10 g/L to about 12 g/L, or about I I g/L to about 12 g/L. 5 [0010a] According to a second aspect of the invention there is provided a recombinant CRM 197 protein produced by the method as described herein. 100111 The present invention relates to a method for producing a recombinant CRM 197 protein in a Pseudomonas host cell, said method comprising: ligating into an expression vector a nucleotide sequence encoding a CRM 197 protein fused to a secretion signal that directs 10 transfer of the CRM 197 protein to the periplasm; transforming the Pseudomonas host cell with the expression vector; and culturing the transformed Pseudomonas host cell in a culture media suitable for the expression of the recombinant CRM 197 protein; wherein the yield of soluble CRM 197 obtained is about I to about 12 grams per liter, and further omprising measuring the activity of the recombinant CRM 197 protein in an activity assay, wherein 15 about 40% to about 100% of the soluble CRM 197 produced is determined to be active. In related embodiments, the activity assay is an immunological assay or a receptor-binding assay. [00121 In embodiments, the expression vector comprises a lac derivative promoter operatively linked to the protein coding sequence, and wherein the culturing comprises induction of the 20 promoter using IPTG at a concentration of about 0.02 to about 1.0 mM, the cell density at induction is an optical density of about 40 to about 200 absorbance units (AU), the pH of the culture is from about 6 to about 7.5, and the growth temperature is about 20 to about 35 'C. 10013] In certain embodiments, the host cell is Pseudomonasfluorescens. 10013a] Unless the context clearly requires otherwise, throughout the description and the claims, the 25 words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to". INCORPORATION BY REFERENCE 100141 All publications, patents, and patent applications mentioned in this specification are herein 30 incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. 5 BRIEF DESCRIPTION OF THE DRAWINGS 100151 The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in 5 which the principles of the invention are utilized, and the accompanying drawings. [00161 Figure 1. Amino Acid and DNA Sequences of an Exemplary Optimized CRM197 Gene. A. Amino acid sequence (SEQ ID NO:I). B. DNA sequence (SEQ ID NO:2). 100171 Figure 2. High Throughput Expression Analysis of CRM197. CRM 197 protein expressed using the DNA sequence shown in Figure l B was analyzed using capillary gel 10 electrophoresis (SDS-CGE). Soluble fractions of 40 CRM 197-expression strains tested are shown in a gel-like image generated from the SDS-CGE data. Strain names as described in Table 6 are listed above each lane. P. fluorescens-expressed CRM 197 migrated as a single band at -58 kDa on SDS-CGE (arrow). DETAILED DESCRIPTION OF THE INVENTION 15 CRM197 100181 Cross-reacting material 197 (CRM 197) is a diphtheria toxin variant produced from a DT gene having a missense mutation. CRM 197 lacks ADP-ribosyltransferase (ADPRT) activity, and is thus nontoxic. The gene for CRM 197 has a single base substitution, resulting in the incorporation of glutamic acid instead of glycine at residue 52. (See, e.g., 20 Bishai, et al., 1987, "High-Level Expression of a Proteolytically Sensitive Diphtheria Toxin Fragment in Escherichia coli," J. Bact. 169(11 ):5 140-51, Giannini, et al., 1984, "The Amino-Acid Sequence of Two Non-Toxic Mutants of Diphtheria Toxin: CRM45 and CRM 197," Nucleic Acids Research 12(10): 4063-9, and GenBank Acc. No. 1007216A, all incorporated herein by reference.) 25 100191 CRM 197 protein may be prepared at low levels by methods known in the art or by expression in C. diphtheriae or other microorganisms. The naturally occurring, or wild type, diphtheria toxin may be obtained from toxin producing strains available from a variety of public sources including the American Type Culture Collection. A plasmid system for producing CRM 197 protein in C. diphtheriae is described by, e.g., U.S. Pat. No. 5,614, 382, 30 "Plasmid for Production of CRM Protein and Diphtheria Toxin," incorporated herein by reference in its entirety. 6 100201 The nucleotide sequence may be prepared using the techniques of recombinant DNA technology (described by, e.g., Sambrook et al, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989), and also by site-directed mutagenesis, based on the known DT nucleotide sequence of the wild type structural gene for diphtheria toxin 5 carried by corynebacteriophage P. (See, e.g., Greenfield, et al., 1993, "Nucleotide Sequence of the Structural Gene for Diphtheria Toxin Carried by Corynebacteriophage 18," Proc Nat Acad Sci 80:6953-7, incorporated herein by reference.) The nucleotide sequence can be optimized as described elsewhere herein. Codon Optimization 10 100211 In heterologous expression systems, optimization steps may improve the ability of the host to produce the foreign protein. Protein expression is governed by a host of factors including those that affect transcription, mRNA processing, and stability and initiation of translation. The polynucleotide optimization steps may include steps to improve the ability of the host to produce the foreign protein as well as steps to assist the researcher in efficiently designing 15 expression constructs. Optimization strategies may include, for example, the modification of translation initiation regions, alteration of mRNA structural elements, and the use of different codon biases. Methods for optimizing the nucleic acid sequence of to improve expression of a heterologous protein in a bacterial host are known in the art and described in the literature. For example, optimization of codons for expression in a Pseudomonas host 20 strain is described, e.g., in U.S. Pat. App. Pub. No.2007/0292918, "Codon Optimization Method," incorporated herein by reference in its entirety. 100221 Optimization can thus address any of a number of sequence features of the heterologous gene. As a specific example, a rare codon-induced translational pause can result in reduced heterologous protein expression. A rare codon-induced translational pause includes the 25 presence of codons in the polynucleotide of interest that are rarely used in the host organism may have a negative effect on protein translation due to their scarcity in the available tRNA pool. One method of improving optimal translation in the host organism includes performing codon optimization which can result in rare host codons being removed from the synthetic polynucleotide sequence. 30 100231 Alternate translational initiation also can result in reduced heterologous protein expression. Alternate translational initiation can include a synthetic polynucleotide sequence inadvertently containing motifs capable of functioning as a ribosome binding site (RBS). These sites can result in initiating translation of a truncated protein from a gene-internal site. 7 One method of reducing the possibility of producing a truncated protein, which can be difficult to remove during purification, includes eliminating putative internal RBS sequences from an optimized polynucleotide sequence. [00241 Repeat-induced polymerase slippage can result in reduced heterologous protein expression. 5 Repeat-induced polymerase slippage involves nucleotide sequence repeats that have been shown to cause slippage or stuttering of DNA polymerase which can result in frameshift mutations. Such repeats can also cause slippage of RNA polymerase. In an organism with a high G+C content bias, there can be a higher degree of repeats composed of G or C nucleotide repeats. Therefore, one method of reducing the possibility of inducing RNA 10 polymerase slippage, includes altering extended repeats of G or C nucleotides. 100251 Interfering secondary structures also can result in reduced heterologous protein expression. Secondary structures can sequester the RBS sequence or initiation codon and have been correlated to a reduction in protein expression. Stemloop structures can also be involved in transcriptional pausing and attenuation. An optimized polynucleotide sequence can contain 15 minimal secondary structures in the RBS and gene coding regions of the nucleotide sequence to allow for improved transcription and translation. 100261 Another feature that can effect heterologous protein expression is the presence of restriction sites. By removing restriction sites that could interfere with subsequent sub-cloning of transcription units into host expression vectors a polynucleotide sequence can be optimized. 20 [00271 For example, the optimization process can begin by identifying the desired amino acid sequence to be heterologously expressed by the host. From the amino acid sequence a candidate polynucleotide or DNA sequence can be designed. During the design of the synthetic DNA sequence, the frequency of codon usage can be compared to the codon usage of the host expression organism and rare host codons can be removed from the synthetic 25 sequence. Additionally, the synthetic candidate DNA sequence can be modified in order to remove undesirable enzyme restriction sites and add or remove any desired signal sequences, linkers or untranslated regions. The synthetic DNA sequence can be analyzed for the presence of secondary structure that may interfere with the translation process, such as G/C repeats and stem-loop structures. Before the candidate DNA sequence is 30 synthesized, the optimized sequence design can be checked to verify that the sequence correctly encodes the desired amino acid sequence. Finally, the candidate DNA sequence can be synthesized using DNA synthesis techniques, such as those known in the art. 8 100281 In another embodiment of the invention, the general codon usage in a host organism, such as P.fluorescens, can be utilized to optimize the expression of the heterologous polynucleotide sequence. The percentage and distribution of codons that rarely would be considered as preferred for a particular amino acid in the host expression system can be evaluated. Values 5 of 5% and 10% usage can be used as cutoff values for the determination of rare codons. For example, the codons listed in Table I have a calculated occurrence of less than 5% in the P. fluorescens MB214 genome and would be generally avoided in an optimized gene expressed in a P.fluorescens host. Table 1. Codons occurring at less than 5% in P.fluorescens MB214 Amino Acid(s) Codon(s) Used % Occurrence G Gly GGA 3.26 I Ile ATA 3.05 L Leu CTA 1.78 CTT 4.57 TTA 1.89 R Arg AGA 1.39 AGG 2.72 CGA 4.99 S Ser TCT 4.28 10 100291 The present invention contemplates the use of any CRM 197 coding sequence, including any sequence that has been optimized for expression in the Pseudomonas host cell being used. Sequences contemplated for use can be optimized to any degree as desired, including, but not limited to, optimization to eliminate: codons occurring at less than 5% in the Pseudomonas host cell, codons occurring at less than 10% in the Pseudomonas host cell, a 15 rare codon-induced translational pause, a putative internal RBS sequence, an extended repeat of G or C nucleotides, an interfering secondary structure, a restriction site, or combinations thereof. 10029a] In embodiments, the amino acid sequence of a secretion leader useful in practicing the methods of the present invention is encoded by any appropriate nucleic acid sequence. 20 Expression Systems 100301 Methods for expressing heterologous proteins, including useful regulatory sequences (e.g., promoters, secretion leaders, and ribosome binding sites), in Pseudomonas host cells, as well as host cells useful in the methods of the present invention, are described, e.g., in U.S. Pat. App. Pub. No. 2008/0269070 and U.S. Pat. App. Ser. No. 12/610,207, both titled 9 "Method for Rapidly Screening Microbial Hosts to Identify Certain Strains with Improved Yield and/or Quality in the Expression of Heterologous Proteins," U.S. Pat. App. Pub. No. 2006/0040352, "Expression of Mammalian Proteins in Pseudomonas Fluorescens," and U.S. Pat. App. Pub. No. 2006/0110747, "Process for Improved Protein Expression by Strain 5 Engineering," all incorporated herein by reference in their entirety. These publications also describe bacterial host strains useful in practicing the methods of the invention, that have been engineered to overexpress folding modulators or wherein protease mutations, including deletions, have been introduced, in order to increase heterologous protein expression. Leaders 10 100311 Sequence leaders are described in detail in U.S. Patent App. Pub. Nos. 2008/0193974 and 2010/0048864, both titled, "Bacterial Leader Sequences for Increased Expression," and U.S. Pat. App. Pub. No. 2006/0008877, "Expression systems with Sec-secretion," all incorporated herein by reference in their entirety, as well as in U.S. Pat. App. Pub. No. 2008/0269070 and U.S. Pat. App. Ser. No. 12/610,207. 15 Table 2. Exemplary Secretion Leader Sequences Secretion Leader Amino Acid Sequence SEQ ID NO: DsbA MRNLILSAALVTASLFGMTAQA 3 Azu MFAKLVAVSLLTLASGQLLA 4 lbp-S3IA MIRDNRLKTSLLRGLTLTLLSLTLLSPAAHA 5 Tpr MNRSSALLLAFVFLSGCQAMA 6 CupB2 MLFRTLLASLTFAVIAGLPSTAHA 7 CupA2 MSCTRAFKPLLLIGLATLMCSHAFA 8 NikA MRLAALPLLLAPLFIAPMAVA 9 Pbp A20V MKLKRLMAAMTFVAAGVATVNAVA 10 DsbC MRLTQIIAAAAIALVSTFALA 11 ToIB MRNLLRGMLVVICCMAGIAAA 12 Pbp MKLKRLMAAMTFVAAGVATANAVA 13 Lao MQNYKKFLLAAAVSMAFSATAMA 14 CupC2 MPPRSIAACLGLLGLLMATQAAA 15 PorE MKKSTLAVAVTLGAIAQQAGA 16 Pbp MKLKRLMAAMTFVAAGVATANAVA 17 FIgI MKFKQLMAMALLLALSAVAQA 18 ttg2C MQNRTVEIGVGLFLLAGILALLLLALRVSGLSA 19 CRM 197 native MSRKLFASXLIGALLGIGAPPSAHA 20 leader 100321 It is understood that the secretion leaders useful in the methods of the present invention are not limited to those disclosed in Table 2. 10 100331 In embodiments, the secretion leader is Azu, lbpS3 1 A, CupA2, or PbpA20V. In other embodiments, the secretion leader is Azu, IbpS3 IA, CupA2, PbpA20V, or Pbp. 100341 Native CR M 197 is transported from C. diptheriae to the extracellular space via a secretion leader that is cleaved, leaving an amino terminal sequence of GADD. In order to preserve 5 the natural amino terminus of CRM 197 following expression in P.fluorescens and ensure disulfide bond formation, the protein is targeted to the periplasmic space. Promoters 100351 Native CRM 197 is transported from C. diptheriae to the extracellular space via a secretion leader that is cleaved, leaving an amino acid sequence of GADD (SEQ ID NO: 21). In order 10 to preserve the natural amino terminus of CRM 197 following expression in P.fluorescens and ensure disulfide bond formation, the protein is targeted to periplasmic space. The promoters used in accordance with the present invention may be constitutive promoters or regulated promoters. Common examples of useful regulated promoters include those of the family derived from the lac promoter (i.e. the lacZ promoter), especially the tac and trc 15 promoters described in U.S. Pat. No. 4,551,433 to DeBoer, as well as Ptacl6, Ptacl7, PtacIl, PlacUV5, and the T71ac promoter. In one embodiment, the promoter is not derived from the host cell organism. In certain embodiments, the promoter is derived from an E. coli organism. 100361 Inducible promoter sequences can be used to regulate expression of CRM 197 in accordance 20 with the methods of the invention. In embodiments, inducible promoters useful in the methods of the present invention include those of the family derived from the lac promoter (i.e. the lacZ promoter), especially the tac and trc promoters described in U.S. Pat. No. 4,551,433 to DeBoer, as well as Ptacl6, Ptacl7, PtacIl, PlacUV5, and the T71ac promoter. In one embodiment, the promoter is not derived from the host cell organism. In certain 25 embodiments, the promoter is derived from an E. coli organism. 10037] Common examples of non-lac-type promoters useful in expression systems according to the present invention include, e.g., those listed in Table 3. Table 3. Examples of non-lac Promoters Promoter Inducer PR High temperature PL High temperature Pm Alkyl- or halo-benzoates I I Pu Alkyl- or halo-toluenes Psal Salicylates 100381 See, e.g.: J. Sanchez-Romero & V. De Lorenzo (1999) Manual of Industrial Microbiology and Biotechnology (A. Demain & J. Davies, eds.) pp. 460-74 (ASM Press, Washington, D.C.); H. Schweizer (2001) Current Opinion in Biotechnology, 12:439-445; and R. Slater & R. Williams (2000 Molecular Biology and Biotechnology (J. Walker & R. Rapley, eds.) pp. 5 125-54 (The Royal Society of Chemistry, Cambridge, UK)). A promoter having the nucleotide sequence of a promoter native to the selected bacterial host cell also may be used to control expression of the transgene encoding the target polypeptide, e.g, a Pseudomonas anthranilate or benzoate operon promoter (Pant, Pben). Tandem promoters may also be used in which more than one promoter is covalently attached to another, whether the same or 10 different in sequence, e.g., a Pant-Pben tandem promoter (interpromoter hybrid) or a Plac Plac tandem promoter, or whether derived from the same or different organisms. 100391 Regulated promoters utilize promoter regulatory proteins in order to control transcription of the gene of which the promoter is a part. Where a regulated promoter is used herein, a corresponding promoter regulatory protein will also be part of an expression system I5 according to the present invention. Examples of promoter regulatory proteins include: activator proteins, e.g., E. co/i catabolite activator protein, MalT protein; AraC family transcriptional activators; repressor proteins, e.g., E. coli Lac proteins; and dual-function regulatory proteins, e.g., E. coli NagC protein. Many regulated-promoter/promoter regulatory-protein pairs are known in the art. In one embodiment, the expression construct 20 for the target protein(s) and the heterologous protein of interest are under the control of the same regulatory element. 100401 Promoter regulatory proteins interact with an effector compound, i.e., a compound that reversibly or irreversibly associates with the regulatory protein so as to enable the protein to either release or bind to at least one DNA transcription regulatory region of the gene that is 25 under the control of the promoter, thereby pennitting or blocking the action of a transcriptase enzyme in initiating transcription of the gene. Effector compounds are classified as either inducers or co-repressors, and these compounds include native effector compounds and gratuitous inducer compounds. Many regulated-promoter/promoter regulatory-protein/effector-compound trios are known in the art. Although an effector 30 compound can be used throughout the cell culture or fermentation, in a preferred embodiment in which a regulated promoter is used, after growth of a desired quantity or 12 density of host cell biomass, an appropriate effector compound is added to the culture to directly or indirectly result in expression of the desired gene(s) encoding the protein or polypeptide of interest. 100411 In embodiments wherein a lac family promoter is utilized, a lac/gene can also be present in 5 the system. The lac gene, which is normally a constitutively expressed gene, encodes the Lac repressor protein Lac protein, which binds to the lac operator of lac family promoters. Thus, where a lac family promoter is utilized, the lac/gene can also be included and expressed in the expression system. 10042] Promoter systems useful in Pseudomonas are described in the literature, e.g., in U.S. Pat. 10 App. Pub. No. 2008/0269070, also referenced above. Other Regulatory Elements [0043] In embodiments, soluble proteins are present in either the cytoplasm or periplasm of the cell during production. Secretion leaders useful for targeting proteins are described elsewhere herein, and in U.S. Pat. App. Pub. No. 2008/0193974, U.S. Pat. App. Pub. No. 15 2006/0008877, and in U.S. Pat. App. Ser. No. 12/610,207. 100441 An expression construct useful in practicing the methods of the present invention can include, in addition to the protein coding sequence, the following regulatory elements operably linked thereto: a promoter, a ribosome binding site (RBS), a transcription terminator, and translational start and stop signals. Useful RBSs can be obtained from any 20 of the species useful as host cells in expression systems according to, e.g., U.S. Pat. App. Pub. No. 2008/0269070 and U.S. Pat. App. Ser. No. 12/610,207. Many specific and a variety of consensus RBSs are known, e.g., those described in and 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 (December 2001 ). In addition, either native or synthetic RBSs may be used, 25 e.g., those described in: EP 0207459 (synthetic RBSs); 0. Ikehata et al., Eur. J. Biochem. 181 (3):563-70 (1989) (native RBS sequence of AAGGAAG). Further examples of methods, vectors, and translation and transcription elements, and other elements useful in the present invention are well known in the art and described in, e.g.: U.S. Pat. No. 5,055,294 to Gilroy and U.S. Pat. No. 5,128,130 to Gilroy et al.; U.S. Pat. No. 5,281,532 to 30 Rammler et al.; U.S. Pat. Nos. 4,695,455 and 4,861,595 to Barnes et al.; U.S. Pat. No. 4,755,465 to Gray et al.; and U.S. Pat. No. 5,169,760 to Wilcox, all incorporated herein by reference, as well as in many of the other publications incorporated herein by reference. 13 Host Strains [00451 Bacterial hosts, including Pseudomonas, and closely related bacterial organisms are contemplated for use in practicing the methods of the invention. In certain embodiments, 5 the Pseudomonas host cell is Pseudomonasfluorescens. The host cell can also be an E. coli cell. [0046] Pseudomonas and closely related bacteria are generally part of the group defined as "Gram(-) Proteobacteria Subgroup I" or "Gram-Negative Aerobic Rods and Cocci" (Buchanan and Gibbons (eds.) (1974) Bergey's Manual of Determinative Bacteriology, pp. 10 217-289). Pseudomonas host strains are described in the literature, e.g., in U.S. Pat. App. Pub. No. 2006/0040352, cited above. 100471 For example, Pseudomonas hosts can include cells from the genus Pseudomonas, Pseudomonas enalia (ATCC 14393), Pseudomonas nigrifaciensi (ATCC 19375), and Pseudomonasputrefaciens (ATCC 8071), which have been reclassified respectively as 1 5 Alleromonas haloplanktis, A lteromonas nigrifaciens, and A 1teromonas putrefaciens. Similarly, e.g., Pseudomonas acidovorans (ATCC 15668) and Pseudomonas tesiosteroni (ATCC 11996) have since been reclassified as Comamonas acidovorans and Comamonas testosteroni, respectively; and Pseudomonas nigrifaciens (ATCC 19375) and Pseudomonas piscicida (ATCC 15057) have been reclassified respectively as Pseudoalteromonas 20 nigrifaciens and Pseudoalteromonas piscicida. 100481 The host cell can be selected from "Gram-negative Proteobacteria Subgroup 16." "Gram negative Proteobacteria Subgroup 16" is defined as the group of Proteobacteria of the following Pseudomonas species (with the ATCC or other deposit numbers of exemplary strain(s) shown in parenthesis): Pseudomonas abietaniphila (ATCC 700689); Pseudomonas 25 aeruginosa (ATCC 10 145); Pseudomonas alcaligenes (ATCC 14909); Pseudomonas anguilliseptica (ATCC 33660); Pseudomonas citronellolis (ATCC 13674); Pseudomonas flavescens (ATCC 51555); Pseudomonas mendocina (ATCC 25411); Pseudomonas nitroreducens (ATCC 33634); Pseudomonas oleovorans (ATCC 8062); Pseudomonas pseudoalcaligenes (ATCC 17440); Pseudomonas resinovorans (ATCC 14235); 30 Pseudomonas straminea (ATCC 33636); Pseudomonas agarici (ATCC 2594 1); Pseudomonas alcaliphila; Pseudomonas alginovora; Pseudomonas andersonii ; Pseudomonas asplenii (ATCC 23835); Pseudomonas azelaica (ATCC 27162); 14 Pseudomonas beyerinckii (ATCC 19372); Pseudomonas borealis; Pseudomonas boreopolis (ATCC 33662); Pseudomonas brassicacearum; Pseudomonas butanovora (ATCC 43655); Pseudomonas cellulosa (ATCC 55703); Pseudomonas aurantiaca (ATCC 33663); Pseudomonas chlororaphis (ATCC 9446, ATCC 13985, ATCC 17418, ATCC 17461); 5 Pseudomonasfragi (ATCC 4973); Pseudomonas lundensis (ATCC 49968); Pseudomonas taetrolens (ATCC 4683); Pseudomonas cissicola (ATCC 33616); Pseudomonas coronafaciens; Pseudomonas diterpeniphila; Pseudomonas elongata (ATCC 10 144); Pseudomonasflectens (ATCC 12775); Pseudomonas azotoformans; Pseudomonas brenneri; Pseudomonas cedrella; Pseudomonas corrugata (ATCC 29736); Pseudomonas 10 extremorientalis; Pseudomonasfluorescens (ATCC 35858); Pseudomonas gessardii; Pseudomonas libanensis; Pseudomonas mandeiji (ATCC 700871); Pseudomonas marginalis (ATCC 10844); Pseudomonas migulae; Pseudomonas mucidolens (ATCC 4685); Pseudomonas orientalis; Pseudomonas rhodesiae; Pseudomonas synxantha (ATCC 9890); Pseudomonas tolaasii (ATCC 33618); Pseudomonas veronii (ATCC 700474); I5 Pseudomonasfrederiksbergensis; Pseudomonas geniculata (ATCC 1 9374); Pseudomonas gingeri; Pseudomonas graminis; Pseudomonas grimontii; Pseudomonas halodenitrificans; Pseudomonas halophila; Pseudomonas hibiscicola (ATCC 19867); Pseudomonas huttiensis (ATCC 14670); Pseudomonas hydrogenovora; Pseudomonasjessenii (ATCC 700870); Pseudomonas kilonensis; Pseudomonas lanceolata (ATCC 14669); Pseudomonas lini; 20 Pseudomonas marginala (ATCC 25417); Pseudomonas mephitica (ATCC 33665); Pseudomonas denitrificans (ATCC 19244); Pseudomonas pertucinogena (ATCC 190); Pseudomonas pictorum (ATCC 23 328); Pseudomonas psychrophila; Pseudomonasfilva (ATCC 31418); Pseudomonas montelii (ATCC 700476); Pseudomonas mosselii; Pseudomonas oryzihabitans (ATCC 43272); Pseudomonas plecoglossicida (ATCC 25 700383); Pseudomonas putida (ATCC 12633); Pseudomonas reactans; Pseudomonas spinosa (ATCC 14606); Pseudomonas balearica; Pseudomonas luteola (ATCC 43273);. Pseudomonas stutzeri (ATCC 17588); Pseudomonas amygdali (ATCC 33614); Pseudomonas avellanae (ATCC 700331); Pseudomonas caricapapayae (ATCC 33615); Pseudomonas cichorii (ATCC 10857); Pseudomonasficuserectae (ATCC 35104); 30 Pseudomonasfuscovaginae; Pseudomonas meliae (A TCC 33050); Pseudomonas syringe (ATCC 193 10); Pseudomonas viridiflava (ATCC 13223); Pseudomonas thermocarboxydovorans (ATCC 35961); Pseudomonas thermotolerans; Pseudomonas thivervalensis; Pseudomonas vancouverensis (ATCC 700688); Pseudomonas wisconsinensis; and Pseudomonas xiamenensis. 15 100491 The host cell can also be selected from "Gram-negative Proteobacteria Subgroup 17." "Gram-negative Proteobacteria Subgroup 17" is defined as the group of Proteobacteria known in the art as the "fluorescent Pseudomonads" including those belonging, e.g., to the following Pseudomonas species: Pseudomonas azotoformans; Pseudomonas brenneri; 5 Pseudomonas cedrella; Pseudomonas corrugata; Pseudomonas extremoriental is; Pseudomonasfluorescens; Pseudomonas gessardii; Pseudomonas libanensis; Pseudomonas mandelii; Pseudomonas marginalis; Pseudomonas migulae; Pseudomonas mucidolens; Pseudomonas orientalis; Pseudomonas rhodesiae; Pseudomonas synxaniha; Pseudomonas tolaasii; and Pseudomonas veronii. 10 100501 Host cells and constructs useful in practicing the methods of the invention can be identified or made using reagents and methods known in the art and described in the literature, e.g., in U.S. Pat. App. Pub. No. 2009/0325230, "Protein Expression Systems," incorporated herein by reference in its entirety. This publication describes production of a recombinant polypeptide by introduction of a nucleic acid construct into an auxotrophic Pseudomonas 15 fluorescens host cell comprising a chromosomal lac gene insert. The nucleic acid construct comprises a nucleotide sequence encoding the recombinant polypeptide operably linked to a promoter capable of directing expression of the nucleic acid in the host cell, and also comprises a nucleotide sequence encoding an auxotrophic selection marker. The auxotrophic selection marker is a polypeptide that restores prototrophy to the auxotrophic 20 host cell. In embodiments, the cell is auxotrophic for proline, uracil, or combinations thereof. In embodiments, the host cell is derived from MB 10 I (ATCC deposit PTA-7841). U. S. Pat. App. Pub. No. 2009/0325230, "Protein Expression Systems," and in Schneider, et al., 2005, "Auxotrophic markers pyrF and proC can replace antibiotic markers on protein production plasmids in high-cell-density Pseudomonasfluorescens fermentation," 25 Biotechnol. Progress 2 1(2): 343-8, both incorporated herein by reference in their entirety, describe a production host strain auxotrophic for uracil that was constructed by deleting the pyrF gene in strain MB 10 1. The pyrF gene was cloned from strain MB214 (ATCC deposit PTA-7840) to generate a plasmid that can complement the pyrF deletion to restore prototropy. In particular embodiments, a dual pyrF-proC dual auxotrophic selection marker 30 system in a P. fluorescens host cell is used. A pyrF production host strain as described can be used as the background for introducing other desired genomic changes, including those described herein as useful in practicing the methods of the invention. 16 10051] In embodiments, the Pseudomonas host cell is defective in the expression of HsIU, HsIV, Prcl, DegP1, DegP2, AprA, or a combination thereof. In embodiments, the host cell is defective in proteases HsIU, HsIV, Prcl, DegP1, DegP2, and AprA. An example of such a strain is disclosed herein as DCI 100. These proteases are known in the art and described in, 5 e.g., U. S. Pat. App. Pub. No. 2006/0 1 10747. AprA, an extracellular serralysin-type metalloprotease metalloproteinase, is described by, e.g., Maunsell, et al., 2006, "Complex regulation of AprA metalloprotease in Pseudomonasfluorescens Ml 14: evidence for the involvement of iron, the ECF sigma factor, PbrA and pseudobactin MI 14 siderophore, Microbiology 152(Pt 1):29-42, incorporated herein by reference, and in U.S. Patent App. 10 Pub. Nos. 2008/0193974 and 2010/0048864. 100521 In other embodiments, the Pseudomonas host cell overexpresses DsbA, DsbB, DsbC, and DsbD. DsbA, B, C, and D are disulfide bond isomerases, described, e.g., in U.S. Pat. App. Pub. No. 2008/0269070 and U.S. Pat. App. Ser. No. 12/610,207. 100531 In other embodiments, the Pseudomonas host cell is wild-type, i.e., having no protease 15 expression defects and not overexpressing any folding modulator. 100541 A host cell that is defective in the expression of a protease can have any modification that results in a decrease in the normal activity or expression level of that protease relative to a wild-type host. For example, a missense or nonsense mutation can lead to expression of protein that not active, and a gene deletion can result in no protein expression at all. A 20 change in the upstream regulatory region of the gene can result in reduced or no protein expression. Other gene defects can affect translation of the protein. The expression of a protease can also be defective if the activity of a protein needed for processing the protease is defective. 10055] Examples of proteases and folding modulators useful in the methods of the present invention 25 are shown in Tables 4 and 5, respectively. RXF numbers refer to the open reading frame. (See, e.g., U.S. Pat. App. Pub. No. 2008/0269070 and U.S. Pat. App. Ser. No. 12/610,207.) Table 4. P.fluorescens strain M!B214 proteases 17 Class Family RXF Gene Curated Function Location Aspartic Peptidases A8 (signal peptidase II family) RXF05383.2 Lipoprotein signal peptidase (ec Cytoplasmic 3.4.23.36) Membrane A24 (type IV prepilin peptidase family) RXF05379.l type 4 prepilin peptidase pild (ec Cytoplasmic 3.4.99.-) Membrane Cysteine Peptidases C15 (pyroglutamyl peptidase I family) RXF02161.1 Pyrrolidone-carboxylate peptidase Cytoplasmic (ec 3.4.19.3) C40 RXFO 1968.1 invasion-associated protein, P60 Signal peptide RXF04920.1 invasion-associated protein, P60 Cytoplasmic RXF04923.1 phosphatase-associated protein Signal peptide papq C56 (Pfpl endopeptidase family) RXF01816.1 protease I (ec 3.4.-.-) Non-secretory Metallopeptidases MI RXF08773.1 Membrane alanine aminopeptidase Non-secretory (ec 3.4.1 1.2) M3 RXF00561.2 prIC Oligopeptidase A (ec 3.4.24.70) Cytoplasmic RXF04631.2 Zn-dependent oligopeptidases Cytoplasmic M4 (thermolysin family) RX F05113.2 Extracellular metalloprotease Extracellular precursor (ec 3.4.24.-) M41 (FtsH endopeptidase family) RXF05400.2 Cell division protein ftsH (ec Cytoplasmic 3.4.24.-) Membrane MIO RXF04304.1 Serralysin (ec 3.4.24.40) Extracellular RX F04500.1 Serralysin (ec 3.4.24.40) Extracellular RXFO 1590.2 Serralysin (ec 3.4.24.40) Extracellular RXF04497.2 Serralysin (ec 3.4.24.40) Extracellular RXF04495.2 Serralysin (ec 3.4.24.40) Extracellular RXF02796.1 Serralysin (ec 3.4.24.40) Extracellular M14 (carboxypeptidase A family) RXF09091.1 Zinc-carboxypeptidase precursor Cytoplasmic (ec 3.4. 17.-) M16 (pitrilysin family) RXF03441.1 Coenzyme pqq synthesis protein F Non-secretory (ec 3.4.99.-) 18 RX FO1918.1 zinc protease (ec 3.4.99.-) Signal peptide RX FO 1919.1 zinc protease (ec 3.4.99.-) Periplasmic RXF03699.2 processing peptidase (ec 3.4.24.64) Signal peptide M17 (leucyl aminopeptidase family) RX F00285.2 Cytosol aminopeptidase (ec Non-secretory 3.4.1l.1) M 18 RXF07879.1 Aspartyl aminopeptidase (ec Cytoplasmic 3.4.11.21) M20 RXF0081 1.1 dapE Succinyl-diaminopimelate Cytoplasmic desuccinylase (ec 3.5.1.18) RXF04052.2 Xaa-His dipeptidase (ec 3.4.13.3) Signal peptide RXF0 1822.2 Carboxypeptidase G2 precursor (ec Signal peptide 3.4.17.11) RXF09831.2:: N-acyl-L-amino acid Signal peptide RXF04892.1 amidohydrolase (ec 3.5.1.14) M28 (aminopeptidase Y family) RXF03488.2 Alkaline phosphatase isozyme OuterMembrane conversion protein precursor (ec 3.4.11.-) M42 (glutamyl aminopeptidase family) RX F05615.1 Deblocking aminopeptidase (ec Non-secretory 3.4.11.-) M22 RXF05817.1 0-sialoglycoprotein endopeptidase Extracellular (ec 3.4.24.57) RXF03065.2 Glycoprotease protein family Non-secretory M23 RXFO1291.2 Cell wall endopeptidase, family Signal peptide M23/M37 RXF03916.1 Membrane proteins related to Signal peptide metal loendopeptidases RXF09147.2 Cell wall endopeptidase, family Signal peptide M23/M37 M24 RXF04693.1 Methionine aminopeptidase (ec Cytoplasmic 3.4.1 1.I 18) RXF03364.1 Methionine aminopeptidase (ec Non-secretory 3.4.1 1.18) RXF02980.1 Xaa-Pro aminopeptidase (ec Cytoplasmic 3.4.1 1.9) RXF06564.1 Xaa-Pro aminopeptidase (ec Cytoplasmic 3.4.1 I .9) M48 (Ste24 endopeptidase family) RXF05137.1 Heat shock protein HtpX Cytoplasmic Membrane RX F0508 1.1 Zinc metalloprotease (ec 3.4.24.-) Signal peptide 19 M50 (S2P protease family) RX F04692.1 Membrane metalloprotease Cytoplasmic Membrane Serine Peptidases Sl (chymotrypsin family) RXF01250.2 protease do (ec 3.4.21.-) Periplasmic RXF07210.1 protease do (ec 3.4.21.-) Periplasmic S8 (subtilisin family) RXF06755.2 serine protease (ec 3.4.2 1.-) Non-secretory RXF08517.1 serine protease (ec 3.4.21.-) Extracellular RXF08627.2 extracellular serine protease (ec Signal peptide 3.4.21.-) RXF06281.1 Extracellular serine protease Non-secretory precursor (ec 3.4.21 .-) RXF08978.1 extracellular serine protease (ec OuterMembrane 3.4.21.-) RXF06451.1 serine protease (ec 3.4.21.-) Signal peptide S9 (prolyl oligopeptidase family) RXF02003.2 Protease ii (ec 3.4.21.83) Periplasmic RX F00458.2 Hydrolase Non-secretory Si I (D-Ala-D-Ala ca rboxypeptidase A family) RXF04657.2 D-alanyl-D-alanine-endopeptidase Periplasmic (ec 3.4.99.-) RXF00670.1 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.-.-) RXF04960.2 D-alanyl-meso-diaminopimelate Signal peptide endopeptidase (ec 3.4.-.-) S14 (ClpP endopeptidase family) RXF04567.1 cipP atp-dependent Cip protease Non-secretory proteolytic subunit (ec 3.4.21.92) RXF04663.1 cipP atp-dependent Clp protease Cytoplasmic proteolytic subunit (ec 3.4.21.92) S16 (ion protease family) RXF04653.2 atp-dependent protease La (ec Cytoplasmic 3.4.21.53) RXF08653.1 atp-dependent protease La (ec Cytoplasmic 3.4.21.53) RXF05943.1 atp-dependent protease La (ec Cytoplasmic 3.4.21.53) S24 (LexA family) RXF00449.1 LexA repressor (ec 3.4.21.88) Non-secretory RXF03397.l LexA repressor (ec 3.4.21.88) Cytoplasmic 20 S26 (signal peptidase I family) RXF01 181.1 Signal peptidase I (ec 3.4.21.89) Cytoplasmic Membrane S33 RXF05236.1 pip3 Proline iminopeptidase (ec 3.4.11.5) Non-secretory RXF04802.l pipl Proline iminopeptidase (ec 3.4.11 .5) Non-secretory RXF04808.2 pip2 Proline iminopeptidase (ec 3.4.11.5) Cytoplasmic S41 (C-terminal processing peptidase family) RXF06586.1 Tail-speci fic protease (ec 3.4.21.-) Signal peptide RXF01037.1 Tail-specific protease (ec 3.4.21.-) Signal peptide S45 RXF07170.1 pacB Penicillin acylase (ec 3.5.1.11) Signal peptide 2 RXF06399.2 pacB Penicillin acylase ii (ec 3.5.1.11) Signal peptide S49 (protease IV family) RXF06993.2 possible protease sohb (ec 3.4.-.-) Non-secretory RXF0 1418.1 protease iv (ec 3.4.-.-) Non-secretory S58 (DmpA aminopeptidase family) RXF06308.2 D-aminopeptidase (ec 3.4.11.19) Cytoplasmic Membrane Threonine Peptidases TI (proteasome family) RX FO 1961.2 hslV atp-dependent protease hslV (ec Cytoplasmic 3.4.25.-) T3 (gam ma-glutamyltransferase family) RXF02342.1 ggtl Gamma-glutamyltranspeptidase (ec Periplasmic 2.3.2.2) RXF04424.2 ggt2 Gamma-glutamyltranspeptidase (ec Periplasmic 2.3.2.2) Unclassified Peptidases U32 RXF00428.1 protease (ec 3.4.-.-) Cytoplasmic RXF02151.2 protease (ec 3.4.-.-) Cytoplasmic U61 RX F04715.1 Muramoyltetrapeptide Non-secretory carboxypeptidase (ec 3.4.17.13) U62 RXF04971.2 pmbA PmbA protein Cytoplasmic RXF04968.2 TldD protein Cytoplasmic Non M EROPS Proteases RXF00325.1 Repressor protein C2 Non-secretory RXF02689.2 Microsomal dipeptidase (ec Cytoplasmic 3.4.13.19) RXF02739.1 membrane dipeptidase (3.4.13.19) Signal peptide RXF03329.2 Hypothetical Cytosolic Protein Cytoplasmic 21 RXF02492.1 Xaa-Pro dipeptidase (ec 3.4.13.9) Cytoplasmic RXF04047.2 caax amino terminal protease Cytoplasmic family Membrane RX F08136.2 protease (transglutaminase-like Cytoplasmic protein) RXF09487.1 Zinc metalloprotease (ec 3.4.24.-) Non-secretory [00561 Certain proteases can have both protease and chaperone-like activity. When these proteases are negatively affecting protein yield and/or quality it can be useful to delete them, and they 5 can be overexpressed when their chaperone activity may positively affect protein yield and/or quality. These proteases include, but are not limited to: Hsp1OO(Clp/Hsl) family members RXF04587.1 (cIpA), RXF08347.1, RXF04654.2 (clpX), RXF04663.1, RXFO 1957.2 (hslU), RXFO I961.2 (hslV); Peptidyl-prolyl cis-trans isomerase family member RXF05345.2 (ppiB); Metallopeptidase M20 family member RXF04892.1 10 (aminohydrolase); Metallopeptidase M24 family members RXF04693.1 (methionine aminopeptidase) and RXF03364.1 (methionine aminopeptidase); and Serine Peptidase S26 signal peptidase I family member RXFO1181.1 (signal peptidase). Table 5. P.fluorescens strain MB214 protein folding modulators ORF ID GENE FUNCTION FAMILY LOCATION GroES/EL RXF02095.1 groES Chaperone HsplO Cytoplasmic RXF06767.l:: groEL Chaperone Hsp60 Cytoplasmic RxfD209O RXF01748.1 ibpA Small heat-shock protein (sHSP) lbpA Hsp20 Cytoplasmic PA3126;Acts as a holder for GroESL folding RXF03385.1 hscB Chaperone protein hscB Hsp20 Cytoplasmic Hsp70 (DnaK/.J) RXF05399.l dnaK Chaperone Hsp70 Periplasmic RXF06954.1 dnaK Chaperone Hsp70 Cytoplasmic RXF03376.1 hscA Chaperone Hsp70 Cytoplasmic RXF03987.2 cbpA Curved dna-binding protein, dnal like activity Hsp40 Cytoplasmic RXF05406.2 dnaJ Chaperone protein dnai Hsp40 Cytoplasmic RXF03346.2 dnaJ Molecular chaperones (DnaJ family) Hsp40 Non-secretory RXF05413.1 grpE heat shock protein GrpE PA4762 GrpE Cytoplasmic Hsp1100 (CP/Hsl) RXF04587.l cIpA atp-dependent clp protease atp-binding subunit HspOO Cytoplasmic clpA RXF08347.1 clpB CIpB protein Hsp 100 Cytoplasmic 22 RXF04654.2 cIpX atp-dependent clp protease atp-binding subunit Hsp100 Cytoplasmic cIpX RX F04663.1 cipP atp-dependent Cip protease proteolytic subunit MEROPS Cytoplasmic (ec 3.4.21.92) peptidase family S 14 RX F01957.2 hslU atp-dependent hsl protease atp-binding subunit Hsp100 Cytoplasmic hslU RXFO1961.2 hsIV atp-dependent hsl protease proteolytic subunit MEROPS Cytoplasmic peptidase subfamily TI B HsP33 RXF04254.2 yrfl 33 kDa chaperonin (Heat shock protein 33 Hsp33 Cytoplasmic homolog) (HSP33). HsP90 RXF05455.2 htpG Chaperone protein htpG Hsp90 Cytoplasmic SecB RX F0223 I .1 secB secretion specific chaperone SecB SecB Non-secretory Disulfide Bond Isomerases RXF07017.2 dsbA disulfide isomerase DSBA oxido- Cytoplasmic reductase RXF08657.2 dsbA/ disulfide isomerase DSBA oxido- Cytoplasmic dsbC/ reductase dsbG/ fernA RX FO1002.1 dsbA/ disulfide isomerase DSBA oxido- Periplasmic dsbC reductase/ Thioredoxin RX F03307.1 dsbC disulfide isomerase Glutaredoxin/ Periplasmic Thioredoxin RX F04890.2 dsbG disulfide isomerase Glutaredoxin/ Periplasmic Thioredoxin RXF03204.1 dsbB Disulfide bond formation protein B (Disulfide DSBA oxido- Periplasmic oxidoreductase). reductase RXF04886.2 dsbD Thiol:disulfide interchange protein dsbD DSBA oxido- Periplasmic reductase Peptidyl-prolyl cis-trans isomerases RXF03768.1 ppiA Peptidyl-prolyl cis-trans isomerase A (ec 5.2.1.8) PPlase: Periplasmic cyclophilin type RXF05345.2 ppiB Peptidyl-prolyl cis-trans isomerase B. PPlase: Cytoplasmic cyclophilin type RXF06034.2 fklB Peptidyl-prolyl cis-trans isomerase FklB. PPlase: OuterMembra FKBP type ne RXF06591.1 fkIB/ fk506 binding protein Peptidyl-prolyl cis-trans PPlase: Periplasmic fkbP isomerase (EC 5.2.1.8) FKBP type 23 RXF05753.2 fklB; Peptidyl-prolyl cis-trans isomerase (ec 5.2.1.8) PPlase: Outer fkbP FKBP type Membrane RXFO1833.2 slyD Peptidyl-prolyl cis-trans isomerase SlyD. PPlase: Non-secretory FKBP type RXF04655.2 tig Trigger factor, ppiase (ec 5.2.1.8) PPlase: Cytoplasmic FKBP type RXF05385.1 yaad Probable FKBP-type 16 kDa peptidyl-prolyl cis- PPlase: Non-secretory trans isomerase (EC 5.2.1.8) (PPiase) FKBP type (Rotamase). RX F00271.1 Peptidyl-prolyl cis-trans isomerase (ec 5.2.1.8) PPlase: Non-secretory FKBP type vili assembly chaperones (pavID like) RXF06068.1 cup Chaperone protein cup pili assembly Periplasmic papD RXF05719.1 ecpD Chaperone protein ecpD pili assembly Signal peptide papD RXF05319.1 ecpD Hnr protein pili assembly Periplasmic chaperone RXF03406.2 ecpD; Chaperone protein ecpD pili assembly Signal peptide csuC papD RXF04296.1 ecpD; Chaperone protein ecpD pili assembly Periplasmic cup papD RXF04553.1 ecpD; Chaperone protein ecpD pili assembly Periplasmic cup papD RXF04554.2 ecpD; Chaperone protein ecpD pili assembly Periplasmic cup papD RXF05310.2 ecpD; Chaperone protein ecpD pili assembly Periplasmic cup papD RXF05304.1 ecpD; Chaperone protein ecpD pili assembly Periplasmic cup papD RXF05073.1 gltF Gram-negative pili assembly chaperone pili assembly Signal peptide periplasmic function papD Type i Secretion Complex RXF05445.1 YacJ H istidinol-phosphate aminotransferase (cc Class-Il Membrane 2.6.1.9) pyridoxal phosphate dependent aminotransfer ase family. Histidinol phosphate aminotransfer ase subfamily. RXF05426.1 SecD Protein translocase subunit secd Type 11 Membrane secretion complex RXF05432.1 SecF protein translocase subunit secf Type il Membrane secretion complex Disulfide Bond Reductases 24 RX F08122.2 trxC Thioredoxin 2 Disulfide Cytoplasmic Bond Reductase RXF0675 1.1 Gor Glutathione reductase (EC 1 .8.1.7) (GR) (GRase) Disulfide Cytoplasmic PA2025 Bond Reductase RX F00922.1 gshA Glutamate--cysteine ligase (ec 6.3.2.2) PA5203 Disulfide Cytoplasmic Bond Reductase Fermentation Format 100571 The expression system according to the present invention can be cultured in any fermentation format. For example, batch, fed-batch, semi-continuous, and continuous 5 fermentation modes may be employed herein. 100581 In embodiments, the fermentation medium may be selected from among rich media, minimal media, and mineral salts media. In other embodiments either a minimal medium or a mineral salts medium is selected. In certain embodiments, a mineral salts medium is selected. 10 100591 Mineral salts media consists of mineral salts and a carbon source such as, e.g., glucose, sucrose, or glycerol. Examples of mineral salts media include, e.g., M9 medium, Pseudomonas medium (ATCC 179), and Davis and Mingioli medium (see, B D Davis & E S Mingioli (1950) J. Bact. 60:17-28). The mineral salts used to make mineral salts media include those selected from among, e.g., potassium phosphates, ammonium sulfate or 15 chloride, magnesium sulfate or chloride, and trace minerals such as calcium chloride, borate, and sulfates of iron, copper, manganese, and zinc. Typically, no organic nitrogen source, such as peptone, tryptone, amino acids, or a yeast extract, is included in a mineral salts medium. Instead, an inorganic nitrogen source is used and this may be selected from among, e.g., ammonium salts, aqueous ammonia, and gaseous ammonia. A mineral salts 20 medium will typically contain glucose or glycerol as the carbon source. In comparison to mineral salts media, minimal media can also contain mineral salts and a carbon source, but can be supplemented with, e.g., low levels of amino acids, vitamins, peptones, or other ingredients, though these are added at very minimal levels. Media can be prepared using the methods described in the art, e.g., in U.S. Pat. App. Pub. No. 2006/0040352, referenced and 25 incorporated by reference above. Details of cultivation procedures and mineral salts media useful in the methods of the present invention are described by Riesenberg, D et al., 1991, "High cell density cultivation of Escherichia coli at controlled specific growth rate," J. Biotechnol. 20 (1):17-27. 25 [00601 In embodiments, production can be achieved in bioreactor cultures. Cultures can be grown in, e.g., up to 2 liter bioreactors containing a mineral salts medium, and maintained at 32 *C and pH 6.5 through the addition of ammonia. Dissolved oxygen can be maintained in excess through increases in agitation and flow of sparged air and oxygen into the fermentor. 5 Glycerol can be delivered to the culture throughout the fermentation to maintain excess levels. In embodiments, these conditions are maintained until a target culture cell density, e.g., optical density at 575nm (As 75 ), for induction is reached, at which time IPTG is added to initiate the target protein production. It is understood that the cell density at induction, the concentration of IPTG, pH and temperature each can be varied to determine optimal 10 conditions for expression. In embodiments, cell density at induction can be varied from A 57 5 of 40 to 200 absorbance units (AU). IPTG concentrations can be varied in the range from 0.02 to 1.0 mM, pH from 6 to 7.5, and temperature from 20 to 35 'C. After 16-24 hours, the culture from each bioreactor can be harvested by centrifugation and the cell pellet frozen at 80 'C. Samples can then be analyzed, e.g., by SDS-CGE, for product formation. 15 10061] Fermentation may be performed at any scale. The expression systems according to the present invention are useful for recombinant protein expression at any scale. Thus, e.g., microliter-scale, milliliter scale, centiliter scale, and deciliter scale fermentation volumes may be used, and I Liter scale and larger fermentation volumes can be used. 100621 In embodiments, the fermentation volume is at or above about I Liter. In embodiments, the 20 fermentation volume is about I liter to about 100 liters. In embodiments, the fermentation volume is about I liter, about 2 liters, about 3 liters, about 4 liters, about 5 liters, about 6 liters, about 7 liters, about 8 liters, about 9 liters, or about 10 liters. In embodiments, the fermentation volume is about I liter to about 5 liters, about I liter to about 10 liters, about I liter to about 25 liters, about I liter to about 50 liters, about I liter to about 75 liters, about 25 10 liters to about 25 liters, about 25 liters to about 50 liters, or about 50 liters to about 100 liters In other embodiments, the fermentation volume is at or above 5 Liters, 10 Liters, 15 Liters, 20 Liters, 25 Liters, 50 Liters, 75 Liters, 100 Liters, 200 Liters, 500 Liters, 1,000 Liters, 2,000 Liters, 5,000 Liters, 10,000 Liters, or 50,000 Liters. Evaluation of Product 30 100631 Numerous assay methods are known in the art for characterizing proteins. Use of any appropriate method for characterizing the yield or quality of the recombinant CRM 197 is contemplated herein. 26 Protein Yield 100641 Protein yield in any purification fraction as described herein can be determined by methods known to those of skill in the art, for example, by capillary gel electrophoresis (CGE), and 5 Western blot analysis. Activity assays, as described herein and known in the art, also can provide information regarding protein yield. 100651 Useful measures of protein yield include, e.g., the amount of recombinant protein per culture volume (e.g., grams or milligrams of protein/liter of culture), percent or fraction of recombinant protein measured in the insoluble pellet obtained after cell lysis (e.g., amount 10 of recombinant protein in extract supernatant/amount of protein in insoluble fraction), percent or fraction of active protein (e.g., amount of active protein/amount protein used in the assay), percent or fraction of total cell protein (tcp), amount of protein/cell, and percent or proportion of dry biomass. 100661 In embodiments wherein yield is expressed in terms of culture volume the culture cell 15 density may be taken into account, particularly when yields between different cultures are being compared. 100671 In embodiments, the methods of the present invention can be used to obtain a recombinant CRM 197 protein yield of about I gram per liter to about 12 grams per liter. In embodiments, the yield is about 0.5 grams per liter to about 12 grams per liter. In certain 20 embodiments, the recombinant protein yield is about 0.5 g/L, about 0.6 g/L, about 0.7 g/L, about 0.8 g/L, about 0.9 g/L, about I g/L, about 1.5 g/L, about 2 g/L, about 2.5 g/L, about 3 g/L, about 3.5 g/L, about 4 g/L, about 4.5 g/L, about 5 g/L, about 5.5 g/L, about 6 g/L, about 6.5 g/L, about 7 g/L, about 7.5 g/L, about 8 g/L, about 8.5 g/L, about 9 g/L, about 9.5 g/L, about 10 g/L, about 10.5 g/L, about I I g/L, about 12 g/L, about 0.5 g/L to about I g/L, 25 about 0.5 g/L to about 2 g/L, about 0.5 g/L to about 3 g/L, about 0.5 g/L to about 4 g/L, about 0.5 g/L to about 5 g/L, about 0.5 g/L to about 6 g/L, about 0.5 g/L to about 7 g/L, about 0.5 g/L to about 8 g/L, about 0.5 g/L to about 9 g/L, about 0.5 g/L to about 10 g/L, about 0.5 g/L to about I I g/L, about 0.5 g/L to about 12 g/L, about I g/L to about 2 g/L, about I g/L to about 3 g/L, about I g/L to about 4 g/L, about I g/L to about 5 g/L, about 1 30 g/L to about 6 g/L, about I g/L to about 7 g/L, about I g/L to about 8 g/L, about I g/L to about 9 g/L, about I g/L to about 10 g/L, about I g/L to about I I g/L, about I g/L to about 12 g/L, about 2 g/L to about 3 g/L, about 2 g/L to about 4 g/L, about 2 g/L to about 5 g/L, 27 about 2 g/L to about 6 g/L, about 2 g/L to about 7 g/L, about 2 g/L to about 8 g/L, about 2 g/L to about 9 g/L, about 2 g/L to about 10 g/L, about 2 g/L to about I I g/L, about 2 g/L to about 12 g/L, about 3 g/L to about 4 g/L, about 3 g/L to about 5 g/L, about 3 g/L to about 6 g/L, about 3 g/L to about 7 g/L, about 3 g/L to about 8 g/L, about 3 g/L to about 9 g/L, about 5 3 g/L to about 10 g/L, about 3 g/L to about I I g/L, about 3 g/L to about 12 g/L, about 4 g/L to about 5 g/L, about 4 g/L to about 6 g/L, about 4 g/L to about 7 g/L, about 4 g/L to about 8 g/L, about 4 g/L to about 9 g/L, about 4 g/L to about 10 g/L, about 4 g/L to about I I g/L, about 4 g/L to about 12 g/L, about 5 g/L to about 6 g/L, about 5 g/L to about 7 g/L, about 5 g/L to about 8 g/L, about 5 g/L to about 9 g/L, about 5 g/L to about 10 g/L, about 5 g/L to 10 about I I g/L, about 5 g/L to about 12 g/L, about 6 g/L to about 7 g/L, about 6 g/L to about 8 g/L, about 6 g/L to about 9 g/L, about 6 g/L to about 10 g/L, about 6 g/L to about 1I g/L, about 6 g/L to about 12 g/L, about 7 g/L to about 8 g/L, about 7 g/L to about 9 g/L, about 7 g/L to about 10 g/L, about 7 g/L to about I I g/L, about 7 g/L to about 12 g/L, about 8 g/L to about 9 g/L, about 8 g/L to about 10 g/L, about 8 g/L to about I 1 g/L, about 8 g/L to about 15 12 g/L, about 9 g/L to about 10 g/L, about 9 g/L to about I I g/L, about 9 g/L to about 12 g/L, about 10 g/L to about I I g/L, about 10 g/L to about 12 g/L, or about I I g/L to about 12 g/L. 10068] In embodiments, the amount of recombinant CRM 197 protein produced is about 1% to 75% of the total cell protein. In certain embodiments, the amount of CRM 197 produced is about 20 1 %, about 2%, about 3%, about 4%, about 5 %, about 10%, about I 5 %, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 1% to about 5%, about 1% to about 10%, about I% to about 20%, about I% to about 30%, about 1% to about 40%, about I% to about 50%, about 1% to about 60%, about 1% to about 75%, about 2% to about 5%, about 2% to about 25 10%, about 2% to about 20%, about 2% to about 30%, about 2% to about 40%, about 2% to about 50%, about 2% to about 60%, about 2% to about 75%, about 3% to about 5%, about 3% to about 10%, about 3% to about 20%, about 3% to about 30%, about 3% to about 40%, about 3% to about 50%, about 3% to about 60%, about 3% to about 75%, about 4% to about 10%, about 4% to about 20%, about 4% to about 30%, about 4% to about 40%, about 4% to 30 about 50%, about 4% to about 60%, about 4% to about 75%, about 5% to about 10%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 60%, about 5% to about 75%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 75%, about 20% to about 30%, about 20% to about 40%, about 20% to 28 about 50%, about 20% to about 60%, about 20% to about 75%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 75%, about 40% to about 50%, about 40% to about 60%, about 40% to about 75%, about 50% to about 60%, about 50% to about 75%, about 60% to about 75%, or about 70% to about 75%, of the total 5 cell protein. Activity 100691 The "solubility" and "activity" of a protein, though related qualities, are generally determined by different means. The solubility of a protein, particularly a hydrophobic protein, typically relates to the folding of a protein; insolubility indicates that hydrophobic 10 amino acid residues are improperly located on the outside of the folded protein. Protein activity, which can be evaluated using methods, e.g., those described below, is another indicator of proper protein conformation. "Soluble, active, or both" as used herein, refers to protein that is determined to be soluble, active, or both soluble and active, by methods known to those of skill in the art. The "activity" of a given protein can include binding 15 activity, e.g., that represented by binding to a receptor, a specific antibody, or to another known substrate, or by enzymatic activity if relevant. Activity levels can be described, e.g., in absolute terms or in relative terms, as when compared with the activity of a standard or control sample, or any sample used as a reference. 100701 Activity assays for evaluating CRM 197 are known in the art and described in the literature. 20 Activity assays include immunological assays, e.g., Western Blot analysis and EL ISA, as well as receptor binding assays, e.g., Diptheria toxin receptor (proHB-EGF) binding. Therefore, a measure of activity can represent, e.g., antibody or receptor binding capacity. 100711 In embodiments, activity is represented by the % active recombinant CRM 197 protein in the extract supernatant as compared with the total amount assayed. This is based on the amount 25 of recombinant CRM 197 protein determined to be active by the assay relative to the total amount of recombinant CRM 197 protein used in the assay. In other embodiments, activity is represented by the % activity level of the protein compared to a standard, e.g., native protein. This is based on the amount of active recombinant CRM 197 protein in supernatant extract sample relative to the amount of active protein in a standard sample (where the same 30 amount of protein from each sample is used in assay). 100721 In embodiments, about 40% to about 100% of the CRM 197 protein is determined to be active. In embodiments, about 40%, about 50%, about 60%, about 70%, about 80%, about 29 90%, or about 100% of the recombinant CRM 197 protein is determined to be active. In embodiments, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 5 100%, about 40% to about 90%, about 40% to about 95%, about 50% to about 90%, about 50% to about 95%, about 50% to about 100%, about 60% to about 90%, about 60% to about 95%, about 60% to about 100%, about 70% to about 90%, about 70% to about 95%, about 70% to about 100%, or about 70% to about 100% of the recombinant CRM 197 protein is determined to be active. 10 100731 In other embodiments, about 75% to about 100% of the recombinant CRM 197 protein is determined to be active. In embodiments, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 80% to about 100%, about 85% to about 90%, about 85% to about 95%, about 85% to about 100%, about 90% to about 95%, about 15 90% to about 100%, or about 95% to about 100% of the recombinant CRM 197 protein is determined to be active. 100741 Means of confirming the identity of the induced protein are also known in the art. For example, a protein can analyzed by peptide mass fingerprint using MALDI-TOF mass spectrometry, N-terminal sequencing analysis, or peptide mapping. 20 100751 While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed 25 in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby. EXAMPLES Example 1: High Throughput Expression of a Recombinant CRM197 Protein 30 100761 CRM197 expression strains were constructed and the amount of soluble CRM 197 protein produced in the strains was analyzed using capillary gel electrophoresis (SDS-CGE). Based on the resulting data, certain strains were selected for use in large-scale expression. 30 Construction and Growth of CRM 197 Expression Strains 100771 The CRM 197 coding sequence was constructed using P. fluorescens preferred codons to encode the CRM 197 amino acid sequence. Figure I shows the amino acid and DNA sequences of the expressed synthetic CRM 197 gene. 5 100781 A standard panel of secretion leaders and host strains was used. Plasmids carrying the codon-optimized CRM 197 sequence, fused to ten P.fluorescens secretion leaders as shown in Table 6, were constructed. The secretion leaders were included to target the protein to the periplasm where for recovery in the properly folded and active form. Table 6. Secretion leaders used for CRM 197 expression screen Lane Secretion Leader DsbA 2 Azu 3 lbp-S31 A 4 Tpr 5 CupB2 6 CupA2 7 NikA 8 Pbp A20V 9 DsbC 10 TolB 10 100791 Constructs containing the ten secretion leaders fused to the recombinant CRM 197 protein were tested in P.fluorescens hosts. Four hosts, listed in Table 7, were tested with each leader. Host cells were electroporated with the indicated plasmids, resuspended in HTP growth medium with trace minerals and 5% glycerol and then transferred to 96-well deep well plate with 400 pl M9 salts I% glucose medium and trace elements. The 96-well plates 15 were incubated at 30*C with shaking for 48 hours. Ten microliters of each of the forty seed cultures were transferred into triplicate 96-well deep-well plates, each well containing 500 .tl of HTP medium supplemented with trace elements and 5% glycerol, and incubated as before for 24 hours. Table 7. Host strains used for CRM 197 expression screen Strain Name Genotype Type DC 1073 Lon-, La-, aprA- PD DC] 100 hslUV-, prcl-, degPl-, degP2-, aprA- PD DC 1125 dsbABCD FMO 31 DC462 grpE, dnaKJ FMO PD = Protease Deletion; FMO = Folding Modulator Overexpressor [00801 Isopropyl-0-D-l-thiogalactopyranoside (IPTG) was added to each well to a final concentration of 0.3 mM to induce the expression of target proteins. Mannitol (Sigma, M 1902) was added to each well to a final concentration of 1% to induce the expression of 5 folding modulators in folding modulator over-expressing strains, and the temperature was reduced to 25*C. Twenty four hours after induction, cells were normalized to OD600 = 15 using PBS in a volume of 400 pl. Samples were frozen for later processing by sonication and centrifugation to generate soluble and insoluble fractions. Sample Preparation and SDS-CGE Analysis 10 100811 Soluble and insoluble cellular fractions were prepared by sonication of the OD-normalized cultures followed by centrifugation. Frozen, normalized culture broth (400 PL) was thawed and sonicated for 3.5 minutes. The lysates were centrifuged at 20,800x g for 20 minutes (4"C) and the supematants removed using manual or automated liquid handling (soluble fraction). The pellets (insoluble fraction) were frozen and then thawed for re-centrifugation 15 at 20,080 x g for 20 minutes at 4 C, to remove residual supernatant. The pellets were then resuspended in 400 pL of I X phosphate buffered saline (PBS), pH 7.4. Further dilutions of soluble and insoluble samples for SDS-CGE analysis were performed in 1 X phosphate buffered saline (PBS), pH 7.4. Soluble and insoluble samples were prepared for SDS capillary gel electrophoresis (CGE) (Caliper Life Sciences, Protein Express LabChip Kit, 20 Part 760301 ), in the presence of dithiothreitol (DTT). [00821 Soluble fractions from each strain expressing target protein were analyzed by reducing SDS-CGE analysis. Representative gel-like images are shown in Figure 2. Table 8 shows the mean soluble CRM 197 yield and standard deviation of 3 replicates for each of the CRM 197-expression strains constructed. The host strain and secretion leader screened for 25 each strain are also indicated. 100831 Both leader and host strain showed a significant impact on CRM 197 expression. Expression ranged from no detectable yield to more than 1.2 g/L at the 0.5mL scale, with the highest expression levels observed in the DCI 100 host background. The yield observed in CS538 746 was 1263 mg/L, and that in CS538-742 was 1241 mg/L, both well over the average 30 yield of 340 mg/L. Both high and low yields were observed in the same host strain 32 depending on the leader used, and both high and low yields were observed using the same leader in different host strains. 100841 CS538-742, CS538-743, CS538-746, CS538-748, CS538-752 were selected for use in large scale fermentation. 5 Table 8. Mean CRM 1 97 yield for CRM 197-expression strains Host Leader Strain Number Man Yield (3 eplies) DC1 073 DsbA CS538-731 205 162 DC1073 Azu CS538-732 427 186 DC1 073 lbp-S31A CS538-733 361 183 DC1073 Tpr CS538-734 298 106 DC1073 CupB2 CS538-735 105 109 DC1 073 CupA2 CS538-736 175 99 DC1073 NikA CS538-737 314 85 DC1073 PbpA20V CS538-738 291 204 DC1 073 DsbC CS538-739 148 91 DC1 073 ToIB CS538-740 213 36 DC1 100 DsbA CS538-741 407 218 DC1 100 Azu CS538-742 1241 372 DC1100 Ibp-S31A cs538-743 1107 219 DC1 100 Tpr CS538-744 280 285 DC1 100 CupB2 CS538-745 192 219 DC1 100 CupA2 CS538-746 1263 474 DC1 100 NikA CS538-747 699 259 DC1 100 PbpA20V CS538-748 914 416 DC1 100 DsbC CS538-749 567 141 DC1 100 ToIB CS538-750 382 217 DC1 125 DsbA CS538-751 591 230 DC1 125 Azu CS538-752 1094 543 DC1 125 lbp-S31A CS538-753 323 143 DC1125 Tpr CS538-754 419 70 DC1 125 CupB2 CS538-755 75 74 DC1 125 CupA2 CS538-756 309 214 DC1 125 NikA CS538-757 52 73 DC1 125 Pbp A20V CS538-758 356 295 DC1125 DsbC CS538-759 319 117 DC1 125 ToIB CS538-760 69 88 DC462 DsbA CS538-761 270 106 DC462 Azu CS538-762 0 14 DC462 lbp-S31A CS538-763 0 6 DC462 Tpr CS538-764 0 0 DC462 CupB2 CS538-765 18 39 DC462 CupA2 CS538-766 118 134 DC462 NikA CS538-767 0 9 33 DC462 Pbp A20V CS538-768 0 0 DC462 DsbC CS538-769 0 0 DC462 TolB CS538-770 0 0 Example 2: Large-scale Expression of a Recombinant CRM197 Protein 100851 Recombinant CRM197 protein is produced in Pseudomonasfluorescens Pfenex Expression TechnologyTM strains CS538-742, CS538-743, CS538-746, CS538-748, CS538-752 in 2 5 liter fermentors. 100861 Cultures are grown in 2 liter fermentors containing a mineral salts medium as described herein and also by, e.g., Riesenberg, D., et al., 1991, and maintained at 32 'C and pH 6.5 through the addition of ammonia. Dissolved oxygen is maintained in excess through increases in agitation and flow of sparged air and oxygen into the fermentor. Glycerol is 10 delivered to the culture throughout the fermentation to maintain excess levels. These conditions are maintained until a target culture cell density (optical density at 575nm (A 5 7 5 )) for induction is reached, at which time IPTG is added to initiate the target protein production. IPTG is added at a concentration of 0.5 mM to initiate CRM 197 production. After 16-24 hours, the culture from each bioreactor is harvested by centrifugation and the 15 cell pellet is frozen at -80 'C. Samples are analyzed by SDS-CGE, for product formation and their activity analyzed by Western Blot. 100871 Yields from large-scale fermentation cultures (e.g., about I liter or more) are typically higher than those obtained in the small HTP cultures. Based on the HTP expression data above, large-scale fermentation yields from about 0.5 to at least 10 g/L are expected. 34