WO2011123139A1 - High level expression of recombinant crm197 - Google Patents

High level expression of recombinant crm197 Download PDF

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WO2011123139A1
WO2011123139A1 PCT/US2010/030573 US2010030573W WO2011123139A1 WO 2011123139 A1 WO2011123139 A1 WO 2011123139A1 US 2010030573 W US2010030573 W US 2010030573W WO 2011123139 A1 WO2011123139 A1 WO 2011123139A1
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pseudomonas
protein
host cell
method
expression
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PCT/US2010/030573
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French (fr)
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Diane M. Retallack
Lawrence Chew
Hongfan Jin
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Pfenex, Inc.
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20Bacteria; Culture media therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/78Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Pseudomonas

Abstract

The present invention relates to the field of recombinant protein production in bacterial hosts. In particular, the present invention relates to a production process for obtaining high levels of a recombinant CRM197 protein from a bacterial host.

Description

HIGH LEVEL EXPRESSION OF RECOMBINANT CRM197

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. application Serial No. 61/319,152, titled '

Level Expression of Recombinant CRM197," filed on March 30, 2010.

BACKGROUND OF THE INVENTION

[0002] Diphtheria toxin (DT) is a proteinaceous toxin that is synthesized and secreted by toxigenic

strains of Corynebacterium diphtheriae. Toxigenic strains contain a bacteriophage lysogen 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 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.

[0003] 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.

diphtheriae that has been infected by the nontoxigenic phage β197ίοχ" created by

nitrosoguanidine mutagenesis of the toxigenic corynephage β. 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.

[0004] 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- 135; Menjugate (Novartis Vaccines), a

meningococcal group C conjugate vaccine; and Prevnar® (Wyeth Pharmaceuticals, Inc.), a childhood pneumonia vaccine that targets seven serotypes of Streptococcus pneumoniae,

and HibTITER® (Wyeth), a Haemophilus influenzae type b vaccine. In addition, CRM 197 has potential use as a boosting antigen for diphtheria and is being investigated as a carrier protein for use in other vaccines.

[0005] A method for high-level expression of CRM 197 for approved therapeutics and

investigational use has not been reported. CRM197 has been expressed in, e.g., C.

diphtheriae, B. subtilis, and E. coli, at levels that range in the tens of mg/L. A single dose of

3934938 l.DOC -1- WSGR Docket No. 38194-734.601 the Prevnar conjugate vaccine contains about 20 μg of CRM197. Therefore, a method for economically producing CRM 197 at levels of about 1 g/L or more would greatly facilitate vaccine research and manufacture.

SUMMARY OF THE INVENTION

[0006] 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

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 1 to about 12 grams per liter.

[0007] 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 hsUJV-, prcl-, degPl-, degP2-, and

aprA-. In embodiments, the the Pseudomonas host cell is hslUV-, prcl-, degPl-, degP2-, and aprA-, and the secretion leader is Azu, IbpS31 A, CupA2, or PbpA20V. In other

embodiments, the Pseudomonas host cell is hslUV-, prcl-, degPl-, degP2-, and aprA-, and the secretion leader is Azu, IbpS31 A, CupA2, PbpA20V, or Pbp. In other embodiments, the

Pseudomonas host cell is defective in the expression of Serralysin, HslU, HslV, Prcl,

DegP 1 , DegP2, or AprA, or the Pseudomonas host cell overexpresses DsbA, DsbB, DsbC, and DsbD.

[0008] 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 host cell is defective in the expression of HslU and HslV, 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.

[0009] In embodiments, the secretion leader is Azu, Pbp, IbpS31 A, CupA2, or PbpA20V. In other

embodiments, the secretion leader is Azu, IbpS31A, CupA2, or PbpA20V.

[0010] In embodiments, the CRM197 nucleotide sequence has been optimized for expression in the

Pseudomonas host cell.

[0011] In embodiments, the yield of soluble CRM197 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 1 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 1 1 g/L, about 12 g/L, about 0.5 g/L

3934938 l.DOC -2- WSGR Docket No. 38194-734.601 to about 1 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

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 1 1 g/L, about 0.5 g/L to about 12 g/L, about 1 g/L to

about 2 g/L, about 1 g/L to about 3 g/L, about 1 g/L to about 4 g/L, about 1 g/L to about 5 g/L, about 1 g/L to about 6 g/L, about 1 g/L to about 7 g/L, about 1 g/L to about 8 g/L, about 1 g/L to about 9 g/L, about 1 g/L to about 10 g/L, about 1 g/L to about 1 1 g/L, about 1 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, 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 1 1 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 3 g/L to about 10 g/L, about 3 g/L to about 1 1 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 1 1 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 about 1 1 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 1 1 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 1 1 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 1 1 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 1 1 g/L, about 9 g/L to about 12 g/L, about 10 g/L to about 1 1 g/L, about 10 g/L to about 12 g/L, or about 1 1 g/L to about 12 g/L.

[0012] 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

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 1 to about 12 grams per liter, and further omprising

measuring the activity of the recombinant CRM 197 protein in an activity assay, wherein

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.

3934938 l.DOC -3- WSGR Docket No. 38194-734.601 [0013] 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 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.

[0014] In certain embodiments, the host cell is Pseudomonas fluorescens.

INCORPORATION BY REFERENCE

[0015] All publications, patents, and patent applications mentioned in this specification are herein

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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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 which the principles of the invention are utilized, and the accompanying

drawings.

[0017] Figure 1. Amino Acid and DNA Sequences of an Exemplary Optimized CRM197

Gene. A. Amino acid sequence (SEQ ID NO: 1). B. DNA sequence (SEQ ID NO:2).

[0018] Figure 2. High Throughput Expression Analysis of CRM197. CRM 197 protein

expressed using the DNA sequence shown in Figure IB was analyzed using capillary gel

electrophoresis (SDS-CGE). Soluble fractions of 40 CRM197-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

CRM 197

[0019] Cross-reacting material 197 (CRM197) 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.,

Bishai, et al., 1987, "High-Level Expression of a Proteolytically Sensitive Diphtheria Toxin

Fragment in Escherichia coli " J. Bact. 169(1 1):5140-51, Giannini, et al., 1984, "The

Amino-Acid Sequence of Two Non-Toxic Mutants of Diphtheria Toxin: CRM45 and

3934938 l.DOC -4- WSGR Docket No. 38194-734.601 CRM197," Nucleic Acids Research 12(10): 4063-9, and GenBank Acc. No. 1007216A, all incorporated herein by reference.)

[0020] 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 CRM197 protein in C. diphtheriae is described by, e.g., U.S. Pat. No. 5,614, 382,

"Plasmid for Production of CRM Protein and Diphtheria Toxin," incorporated herein by

reference in its entirety.

[0021] 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 carried by corynebacteriophage β. (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

[0022] 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 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 strain is described, e.g., in U.S. Pat. App. Pub. No.2007/0292918, "Codon Optimization

Method," incorporated herein by reference in its entirety.

[0023] 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

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

3934938 l.DOC -5- WSGR Docket No. 38194-734.601 performing codon optimization which can result in rare host codons being removed from the synthetic polynucleotide sequence.

[0024] 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.

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.

[0025] Repeat- induced polymerase slippage can result in reduced heterologous protein expression.

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

polymerase slippage, includes altering extended repeats of G or C nucleotides.

[0026] 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 minimal secondary structures in the RBS and gene coding regions of the nucleotide

sequence to allow for improved transcription and translation.

[0027] 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.

[0028] 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

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

synthesized, the optimized sequence design can be checked to verify that the sequence

3934938 l.DOC -6- WSGR Docket No. 38194-734.601 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.

[0029] 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 of 5% and 10% usage can be used as cutoff values for the determination of rare codons. For example, the codons listed in Table 1 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) ( odon(s) ί M d " .. Occu rrence

G Gly GGA 3.26

I He 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

[0030] 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 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.

[0031] Furthermore, the amino acid sequence of any secretion leader useful in practicing the

methods of the present invention can be encoded by any appropriate nucleic acid sequence.

Expression Systems

[0032] 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

"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

3934938 l.DOC -7- WSGR Docket No. 38194-734.601 U.S. Pat. App. Pub. No. 2006/01 10747, "Process for Improved Protein Expression by Strain

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

[0033] 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.

Table 2. Exemplary Secretion Leader Sequences

Si-ci tion Amino Acid Si-qiu-nci' SKQ I I) NO: Ι Λ'ΪΚΙΙΊ·

DsbA MRNLILSAALVTASLFGMTAQA 3

Azu MFAKLVAVSLLTLASGQLLA 4

Ibp-S31A MIRDNRLKTSLLRGLTLTLLSLTLLSPAAIL 5

Tpr MNRSSALLLAFVFLSGCQAMA 6

CupB2 MLFRTLLASLTFAVIAGLPSTAHA 7

CupA2 MSCTRAFKPLLLIGLATLMCSHAFA 8

NikA MRLAALPLLLAPLFIAPMAVA 9

Pbp A20V MKLKRLMAAMTFVAAGVATVNAVA 10

DsbC MRLTQIIAAAAIALVSTFALA 1 1

TolB MRNLLRGMLWICCMAGIAAA 12

Pbp MKLKRLMAAMTFVAAGVATANAVA 13

Lao MQ YKKFLLAAAVSMAF S AT AMA 14

CupC2 MPPRSIAACLGLLGLLMATQAAA 15

PorE MKKSTLAVAVTLGAIAQQAGA 16

Pbp MKLKRLMAAMTFVAAGVATANAVA 17

Flgl MKFKQLMAMALLLALSAVAQA 18

ttg2C A [QNRTVEIGVGLFLLAGILALLLLALRVSGL 3A 19

CRM 197 native MSRKLFASXLIGALLGIGAPPSAHA 20

leader

It is understood that th e secretion leaders useful in the methods of the present invention are not limited to those disclosed in Table 2.

[0035] In embodiments, the secretion leader is Azu, IbpS31 A, CupA2, or PbpA20V. In other

embodiments, the secretion leader is Azu, IbpS31A, CupA2, PbpA20V, or Pbp.

[0036] Native CRM197 is transported from C. diptheriae to the extracellular space via a secretion

leader that is cleaved, leaving an amino terminal sequence of GADD (SEQ ID NO: 21). In order to preserve 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

3934938 l.DOC -8- WSGR Docket No. 38194-734.601 [0037] 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 promoters described in U.S. Pat. No. 4,551 ,433 to DeBoer, as well as Ptacl 6, Ptacl 7, PtacII, 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.

[0038] Inducible promoter sequences can be used to regulate expression of CRM 197 in accordance

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 Ptac l 6, Ptac l7, PtacII, 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.

[0039] 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

l Yomoier I nducer

PR High temperature

PL High temperature

Pm Alkyl- or halo-benzoates

Pu Alkyl- or halo-toluenes

Psal Salicylates

[0040] 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.

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

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.

[0041] 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

3934938 l.DOC -9- WSGR Docket No. 38194-734.601 according to the present invention. Examples of promoter regulatory proteins include: activator proteins, e.g., E. coli catabolite activator protein, MalT protein; AraC family

transcriptional activators; repressor proteins, e.g., E. coli Lacl 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 for the target protein(s) and the heterologous protein of interest are under the control of the same regulatory element.

[0042] 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 under the control of the promoter, thereby permitting 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

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

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.

[0043] In embodiments wherein a lac family promoter is utilized, a lacl gene can also be present in

the system. The lacl gene, which is normally a constitutively expressed gene, encodes the

Lac repressor protein Lacl protein, which binds to the lac operator of lac family promoters.

Thus, where a lac family promoter is utilized, the lacl gene can also be included and

expressed in the expression system.

[0044] Promoter systems useful in Pseudomonas are described in the literature, e.g., in U.S. Pat.

App. Pub. No. 2008/0269070, also referenced above.

Other Regulatory Elements

[0045] 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.

2006/0008877, and in U.S. Pat. App. Ser. No. 12/610,207.

[0046] 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

3934938 l.DOC -10- WSGR Docket No. 38194-734.601 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): 1 123-30 (December 2001). In addition, either native or synthetic RBSs may be used, e.g., those described in: EP 0207459 (synthetic RBSs); O. 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

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.

Host Strains

[0047] Bacterial hosts, including Pseudomonas, and closely related bacterial organisms are

contemplated for use in practicing the methods of the invention. In certain embodiments, the Pseudomonas host cell is Pseudomonas fluorescens. The host cell can also be an E. coli cell.

[0048] Pseudomonas and closely related bacteria are generally part of the group defined as "Gram(- ) Proteobacteria Subgroup 1 " or "Gram-Negative Aerobic Rods and Cocci" (Buchanan and

Gibbons (eds.) (1974) Bergey's Manual of Determinative Bacteriology, pp. 217-289).

Pseudomonas host strains are described in the literature, e.g., in U.S. Pat. App. Pub. No.

2006/0040352, cited above.

[0049] For example, Pseudomonas hosts can include cells from the genus Pseudomonas,

Pseudomonas enalia (ATCC 14393), Pseudomonas nigrifaciensi (ATCC 19375), and

Pseudomonas putrefaciens (ATCC 8071), which have been reclassified respectively as

Alteromonas haloplanktis , Alteromonas nigrifaciens, and Alteromonas putrefaciens.

Similarly, e.g., Pseudomonas acidovorans (ATCC 15668) and Pseudomonas testosteroni

(ATCC 1 1996) 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

nigrifaciens and Pseudoalteromonas piscicida.

[0050] 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

3934938 l.DOC -1 1- WSGR Docket No. 38194-734.601 aeruginosa (ATCC 10145); Pseudomonas alcaligenes (ATCC 14909); Pseudomonas

anguilliseptica (ATCC 33660); Pseudomonas citronellolis (ATCC 13674); Pseudomonas flavescens (ATCC 51555); Pseudomonas mendocina (ATCC 2541 1); Pseudomonas

nitroreducens (ATCC 33634); Pseudomonas oleovorans (ATCC 8062); Pseudomonas

pseudoalcaligenes (ATCC 17440); Pseudomonas resinovorans (ATCC 14235);

Pseudomonas straminea (ATCC 33636); Pseudomonas agarici (ATCC 25941);

Pseudomonas alcaliphila; Pseudomonas alginovora; Pseudomonas andersonii;

Pseudomonas asplenii (ATCC 23835); Pseudomonas azelaica (ATCC 27162);

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);

Pseudomonas fragi (ATCC 4973); Pseudomonas lundensis (ATCC 49968); Pseudomonas taetrolens (ATCC 4683); Pseudomonas cissicola (ATCC 33616); Pseudomonas

coronafaciens; Pseudomonas diterpeniphila; Pseudomonas elongata (ATCC 10144);

Pseudomonasflectens (ATCC 12775); Pseudomonas azotoformans; Pseudomonas brenneri;

Pseudomonas cedrella; Pseudomonas corrugata (ATCC 29736); Pseudomonas

extremorientalis; Pseudomonas fluorescens (ATCC 35858); Pseudomonas gessardii;

Pseudomonas libanensis; Pseudomonas mandelii (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);

Pseudomonas frederiksbergensis; Pseudomonas geniculata (ATCC 19374); Pseudomonas gingeri; Pseudomonas graminis; Pseudomonas grimontii; Pseudomonas halodenitrificans;

Pseudomonas halophila; Pseudomonas hibiscicola (ATCC 19867); Pseudomonas huttiensis

(ATCC 14670); Pseudomonas hydrogenovora; Pseudomonas jessenii (ATCC 700870);

Pseudomonas kilonensis; Pseudomonas lanceolata (ATCC 14669); Pseudomonas lini;

Pseudomonas marginata (ATCC 25417); Pseudomonas mephitica (ATCC 33665);

Pseudomonas denitrificans (ATCC 19244); Pseudomonas pertucinogena (ATCC 190);

Pseudomonas pictorum (ATCC 23328); Pseudomonas psychrophila; Pseudomonas filva

(ATCC 31418); Pseudomonas monteilii (ATCC 700476); Pseudomonas mosselii;

Pseudomonas oryzihabitans (ATCC 43272); Pseudomonas plecoglossicida (ATCC

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); .DOC -12- WSGR Docket No. 38194-734.601 Pseudomonas cichorii (ATCC 10857); Pseudomonas ficuserectae (ATCC 35104);

Pseudomonas fuscovaginae; Pseudomonas meliae (ATCC 33050); Pseudomonas syringae

(ATCC 19310); Pseudomonas viridiflava (ATCC 13223); Pseudomonas

thermocarboxydovorans (ATCC 35961); Pseudomonas thermotolerans; Pseudomonas

thivervalensis; Pseudomonas vancouverensis (ATCC 700688); Pseudomonas

wisconsinensis; and Pseudomonas xiamenensis.

[0051] 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;

Pseudomonas cedrella; Pseudomonas corrugata; Pseudomonas extremorientalis;

Pseudomonas fluorescens; Pseudomonas gessardii; Pseudomonas libanensis; Pseudomonas mandelii; Pseudomonas marginalis; Pseudomonas migulae; Pseudomonas mucidolens;

Pseudomonas orientalis; Pseudomonas rhodesiae; Pseudomonas synxantha; Pseudomonas tolaasii; and Pseudomonas veronii.

[0052] 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

fluorescens host cell comprising a chromosomal lacl 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 host cell. In embodiments, the cell is auxotrophic for proline, uracil, or combinations

thereof. In embodiments, the host cell is derived from MB101 (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 Pseudomonas fluorescens fermentation,"

Biotechnol. Progress 21(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 101. 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 system in a P. fluorescens host cell is used. A pyrF production host strain as described can

3934938 l.DOC -13- WSGR Docket No. 38194-734.601 be used as the background for introducing other desired genomic changes, including those described herein as useful in practicing the methods of the invention.

[0053] In embodiments, the Pseudomonas host cell is defective in the expression of HslU, HslV,

Prcl, DegPl, DegP2, AprA, or a combination thereof. In embodiments, the host cell is

defective in proteases HslU, HslV, Prcl, DegPl, DegP2, and AprA. An example of such a strain is disclosed herein as DC 1100. These proteases are known in the art and described in, e.g., U. S. Pat. App. Pub. No. 2006/01 10747. AprA, an extracellular serralysin-type

metalloprotease metalloproteinase, is described by, e.g., Maunsell, et al., 2006, "Complex regulation of AprA metalloprotease in Pseudomonas fluorescens Ml 14: evidence for the

involvement of iron, the ECF sigma factor, PbrA and pseudobactin Ml 14 siderophore,

Microbiology 152(Pt l):29-42, incorporated herein by reference, and in U.S. Patent App.

Pub. Nos. 2008/0193974 and 2010/0048864.

[0054] 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.

[0055] In other embodiments, the Pseudomonas host cell is wild-type, i.e., having no protease

expression defects and not overexpressing any folding modulator.

[0056] 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

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.

[0057] Examples of proteases and folding modulators useful in the methods of the present

invention 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 MB214 proteases

3934938 l.DOC -14- WSGR Docket No. 38194-734.601

Figure imgf000016_0001

938 l.DOC -15- WSGR Docket No. 38194-734.601 RXF01918.1 zinc protease (ec 3.4.99.-) Signal peptide

RXF01919.1 zinc protease (ec 3.4.99.-) Periplasmic

RXF03699.2 processing peptidase (ec 3.4.24.64) Signal peptide

M17 (leucyl aminopeptidase family)

RXF00285.2 Cytosol aminopeptidase (ec Non- secretory

3.4.11.1)

M18

RXF07879.1 Aspartyl aminopeptidase (ec Cytoplasmic

3.4.11.21)

M20

RXF00811.1 dapE Succinyl-diaminopimelate Cytoplasmic

desuccinylase (ec 3.5.1.18)

RXF 04052.2 Xaa-His dipeptidase (ec 3.4.13.3) Signal peptide

RXF01822.2 Carboxypeptidase G2 precursor (ec Signal peptide

3.4.17.11)

RXF 09831.2:: N-acyl-L-amino acid Signal peptide

RXF 04892.1 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)

RXF05615.1 Deblocking aminopeptidase (ec Non- secretory

3.4.11.-)

M22

RXF05817.1 O - sialoglycoprotein endopeptidase Extracellular

(ec 3.4.24.57)

RXF03065.2 Glycoprotease protein family Non-secretory

M23

RXF01291.2 Cell wall endopeptidase, family Signal peptide

M23/M37

RXF03916.1 Membrane proteins related to Signal peptide

metalloendopeptidases

RXF09147.2 Cell wall endopeptidase, family Signal peptide

M23/M37

M24

RXF04693.1 Methionine aminopeptidase (ec Cytoplasmic

3.4.11.18)

RXF03364.1 Methionine aminopeptidase (ec Non- secretory

3.4.11.18)

RXF02980.1 Xaa-Pro aminopeptidase (ec Cytoplasmic

3.4.11.9)

RXF06564.1 Xaa-Pro aminopeptidase (ec Cytoplasmic

3.4.11.9)

M48 (Ste24 endopeptidase family)

RXF05137.1 Heat shock protein HtpX Cytoplasmic

Membrane

RXF05081.1 Zinc metalloprotease (ec 3.4.24.-) Signal peptide

-16- WSGR Docket No. 38194-734.601 M50 (S2P protease family)

RXF 04692.1 Membrane metalloprotease Cytoplasmic

Membrane

Serine rnl iihi 1**

SI (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 3.4.21.-) 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

RXF00458.2 Hydrolase Non- secretory

Sll (D-Ala-D-Ala carboxypeptidase A family)

RXF 04657.2 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)

RXF00133.1 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 clpP atp-dependent Clp protease Non- secretory

proteolytic subunit (ec 3.4.21.92)

RXF04663.1 clpP atp-dependent Clp protease Cytoplasmic

proteolytic subunit (ec 3.4.21.92)

S16 (Ion protease family)

RXF 04653.2 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

RXF03397.1 LexA repressor (ec 3.4.21.88) Cytoplasmic

938 l.DOC -17- WSGR Docket No. 38194-734.601

Figure imgf000019_0001

4938 l.DOC -18- WSGR Docket No.38194-734.601 RXF02492.1 Xaa-Pro dipeptidase (ec 3.4.13.9) Cytoplasmic

RXF04047.2 caax amino terminal protease Cytoplasmic

family Membrane

RXF08136.2 protease (transglutaminase-like Cytoplasmic

protein)

RXF 09487.1 Zinc metalloprotease (ec 3.4.24.-) Non-secretory

[0058] 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 can be overexpressed when their chaperone activity may positively affect protein yield

and/or quality. These proteases include, but are not limited to: Hsp l00(Clp/Hsl) family

members RXF04587.1 (clpA), RXF08347.1 , RXF04654.2 (clpX), RXF04663.1,

RXF01957.2 (hslU), RXF01961.2 (hslV); Peptidyl-prolyl cis-trans isomerase family

member RXF05345.2 (ppiB); Metallopeptidase M20 family member RXF04892.1

(aminohydrolase); Metallopeptidase M24 family members RXF04693.1 (methionine

ammopeptidase) and RXF03364.1 (methionine ammopeptidase); and Serine Peptidase S26 signal peptidase I family member RXF01 181.1 (signal peptidase).

Table 5. P. fluoresceins strain MB214 protein folding modulators

ORF ID GENE FUNCTION FAMILY LOCATION

GroES/EL

RXF02095.1 groES Chaperone HsplO Cytoplasmic

RXF06767.1 :: groEL Chaperone Hsp60 Cytoplasmic

Rxf02090

RXF01748.1 ibpA Small heat-shock protein (sHSP) IbpA Hsp20 Cytoplasmic

PA3126;Acts as a holder for GroESL folding

RXF03385.1 hscB Chaperone protein hscB Hsp20 Cytoplasmic

HSD70 (OnaK/J)

RXF05399.1 dnaK Chaperone Hsp70 Periplasmic

RXF06954.1 dnaK Chaperone Hsp70 Cytoplasmic

RXF03376.1 hscA Chaperone Hsp70 Cytoplasmic

RXF03987.2 cbpA Curved dna-binding protein, dnaJ like activity Hsp40 Cytoplasmic

RXF05406.2 dnaJ Chaperone protein dnaJ Hsp40 Cytoplasmic

RXF03346.2 dnaJ Molecular chaperone s (DnaJ family) Hsp40 Non- secretory

RXF05413.1 grpE heat shock protein GrpE PA4762 GrpE Cytoplasmic

HSDIOO iClD/Hsl)

RXF04587.1 clpA atp-dependent clp protease atp-binding subunit HsplOO Cytoplasmic

clpA

RXF08347.1 clpB ClpB protein HsplOO Cytoplasmic

3934938 l.DOC - 19- WSGR Docket No. 38194-734.601

Figure imgf000021_0001

FKBP type

3934938 l.DOC -20- WSGR Docket No. 38194-734.601 RXF04655.2 tig Trigger factor, ppiase (ec 5.2.1.8) PPiase: Cytoplasmic

FKBP type

RXF05385.1 yaad Probable FKBP-type 16 kDa peptidyl-prolyl cis- PPiase: Non- secretory

trans isomerase (EC 5.2.1.8) (PPiase) FKBP type

(Rotamase).

RXF00271.1 Peptidyl-prolyl cis-trans isomerase (ec 5.2.1.8) PPiase: Non- secretory

FKBP type pili assembly chaperones (papD 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 II Secretion Complex

RXF05445.1 YacJ Histidinol-phosphate aminotransferase (ec Class-II Membrane

2.6.1.9) pyridoxal- phosphate- dependent

aminotransfer

ase family.

Histidinol- phosphate

aminotransfer

ase

subfamily.

RXF05426.1 SecD Protein translocase subunit seed Type II Membrane

secretion

complex

RXF05432.1 SecF protein translocase subunit secf Type II Membrane

secretion

complex

Disulfide Bond Reductases

RXF08122.2 trxC Thioredoxin 2 Disulfide Cytoplasmic

Bond

Reductase

RXF06751.1 Gor Glutathione reductase (EC 1.8.1.7) (GR) (GRase) Disulfide Cytoplasmic

PA2025 Bond

Reductase

RXF00922.1 gshA Glutamate— cysteine ligase (ec 6.3.2.2) PA5203 Disulfide Cytoplasmic

Bond

3934938 l.DOC -21- WSGR Docket No. 38194-734.601 Reductase

Fermentation Format

[0059] The expression system according to the present invention can be cultured in any

fermentation format. For example, batch, fed-batch, semi-continuous, and continuous

fermentation modes may be employed herein.

[0060] 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.

[0061] 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

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

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 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.

[0062] 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.

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 (A575), 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,

3934938 l.DOC -22- WSGR Docket No. 38194-734.601 the concentration of IPTG, pH and temperature each can be varied to determine optimal

conditions for expression. In embodiments, cell density at induction can be varied from A575 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.

[0063] 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 1 Liter scale and larger fermentation volumes can be used.

[0064] In embodiments, the fermentation volume is at or above about 1 Liter. In embodiments, the

fermentation volume is about 1 liter to about 100 liters. In embodiments, the fermentation volume is about 1 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 1 liter to about 5 liters, about 1 liter to about 10 liters, about 1 liter to about 25 liters, about 1 liter to about 50 liters, about 1 liter to about 75 liters, about

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

[0065] 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.

Protein Yield

[0066] 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

Western blot analysis. Activity assays, as described herein and known in the art, also can

provide information regarding protein yield.

[0067] 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 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.

3934938 l.DOC -23- WSGR Docket No. 38194-734.601 [0068] In embodiments wherein yield is expressed in terms of culture volume the culture cell density may be taken into account, particularly when yields between different cultures are being compared.

[0069] In embodiments, the methods of the present invention can be used to obtain a recombinant

CRM 197 protein yield of about 1 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

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 1 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 1 1 g/L, about 12 g/L, about 0.5 g/L to about 1 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 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 1 1 g/L, about 0.5 g/L to about 12 g/L, about 1 g/L to about 2 g/L,

about 1 g/L to about 3 g/L, about 1 g/L to about 4 g/L, about 1 g/L to about 5 g/L, about 1 g/L to about 6 g/L, about 1 g/L to about 7 g/L, about 1 g/L to about 8 g/L, about 1 g/L to

about 9 g/L, about 1 g/L to about 10 g/L, about 1 g/L to about 1 1 g/L, about 1 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, 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 11 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 3 g/L to about 10 g/L, about 3 g/L to about 1 1 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 1 1 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 about 11 g/L, about 5 g/L to about 12 g/L, about 6 g/L to about 7 g/L, about 6 g/L to about 8 g/L, about 6 g/L to about 9 g/L, about 6 g/L to about 10 g/L, about 6 g/L to about 1 1 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 1 1 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 1 1 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 1 1 g/L, about 9 g/L to about 12

g/L, about 10 g/L to about 1 1 g/L, about 10 g/L to about 12 g/L, or about 1 1 g/L to about 12 g/L.

3934938 l.DOC -24- WSGR Docket No. 38194-734.601 [0070] 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

1%, about 2%, about 3%, about 4%, about 5 %, about 10%, about 15 %, 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

1% to about 20%, about 1% to about 30%, about 1% to about 40%, about 1% to about 50%, about 1% to about 60%, about 1% to about 75%, about 2% to about 5%, about 2% to about

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 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 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 cell protein.

Activity

[0071] 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 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

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.

3934938 l.DOC -25- WSGR Docket No. 38194-734.601 [0072] Activity assays for evaluating CRM 197 are known in the art and described in the literature.

Activity assays include immunological assays, e.g., Western Blot analysis and ELISA, 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.

[0073] 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 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 amount of protein from each sample is used in assay).

[0074] 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

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

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.

[0075] 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

90% to about 100%., or about 95% to about 100%. of the recombinant CRM 197 protein is

determined to be active.

[0076] 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.

[0077] 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

3934938 l.DOC -26- WSGR Docket No. 38194-734.601 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 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

[0078] CRM 197 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.

Construction and Growth of CRM 197 Expression Strains

[0079] The CRM 197 coding sequence was constructed using P. fluorescens preferred codons to

encode the CRM 197 amino acid sequence. Figure 1 shows the amino acid and DNA

sequences of the expressed synthetic CRM 197 gene.

[0080] 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

Figure imgf000028_0001

[0081] Constructs containing the ten secretion leaders fused to the recombinant CRM197 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 μΐ M9 salts 1% glucose medium and trace elements. The 96-well plates 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

3934938 l.DOC -27- WSGR Docket No. 38194-734.601 μΐ 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

Figure imgf000029_0001

PD = Protease Deletion; FMO = Folding Modulator Overexpressor

[0082] Isopropyl^-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,

Ml 902) was added to each well to a final concentration of 1% to induce the expression of 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 μΐ. Samples were frozen for later processing by sonication

and centrifugation to generate soluble and insoluble fractions.

Sample Preparation and SDS-CGE Analysis

[0083] Soluble and insoluble cellular fractions were prepared by sonication of the OD-normalized

cultures followed by centrifugation. Frozen, normalized culture broth (400 μί) was thawed and sonicated for 3.5 minutes. The lysates were centrifuged at 20,800x g for 20 minutes

(4°C) and the supernatants removed using manual or automated liquid handling (soluble

fraction). The pellets (insoluble fraction) were frozen and then thawed for re-centrifugation at 20,080 x g for 20 minutes at 4 C, to remove residual supernatant. The pellets were then resuspended in 400 μΐ^ of IX phosphate buffered saline (PBS), pH 7.4. Further dilutions of soluble and insoluble samples for SDS-CGE analysis were performed in IX 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,

Part 760301), in the presence of dithiothreitol (DTT).

[0084] 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

CRM197-expression strains constructed. The host strain and secretion leader screened for each strain are also indicated.

[0085] Both leader and host strain showed a significant impact on CRM197 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 DC 1 100 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

yield of 340 mg/L. Both high and low yields were observed in the same host strain

3934938 l.DOC -28- WSGR Docket No. 38194-734.601 depending on the leader used, and both high and low yields were observed using the same leader in different host strains.

[0086] CS538-742, CS538-743, CS538-746, CS538-748, CS538-752 were selected for use in large- scale fermentation.

Table 8. Mean CRM197 yield for CRM197-expression strains

Figure imgf000030_0001

Example 2: Large-scale Expression of a Recombinant CRM197 Protein

3934938 l.DOC -29- WSGR Docket No. 38194-734.601 [0087] Recombinant CRM 197 protein is produced in Pseudomonas fluoresceins Pfenex Expression

Technology™ strains CS538-742, CS538-743, CS538-746, CS538-748, CS538-752 in 2

liter fermentors.

[0088] 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

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 (A575)) 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 CRM197 production.

After 16-24 hours, the culture from each bioreactor is harvested by centrifugation and the

cell pellet is frozen at -80 °C. Samples are analyzed by SDS-CGE, for product formation

and their activity analyzed by Western Blot.

[0089] Yields from large-scale fermentation cultures (e.g., about 1 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.

3934938 l.DOC -30- WSGR Docket No. 38194-734.601

Claims

CLAIMS WHAT IS CLAIMED IS:
1. 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; 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.
2. The method of claim 1, wherein the Pseudomonas host cell is defective in the
expression of at least one protease or wherein the Pseudomonas host cell overexpresses at least one folding modulator.
3. The method of claim 2, wherein the Pseudomonas host cell is hslUV-, prcl-, degPl- , degP2-, and aprA-.
4. The method of claim 3, wherein the secretion leader is Azu, IbpS31 A, CupA2,
PbpA20V, or Pbp.
5. The method of claim 2, wherein the Pseudomonas host cell is defective in the
expression of Serralysin, HslU, HslV, Prcl, DegPl, DegP2, or AprA, or wherein the Pseudomonas host cell overexpresses DsbA, DsbB, DsbC, and DsbD.
6. The method of claim 5, wherein the host cell overexpresses DsbA, DsbB, DsbC,
and DsbD, and the secretion leader is Azu.
3934938 l.DOC -31- WSGR Docket No. 38194-734.601
7. The method of claim 5, wherein the host cell is defective in the expression of Serralysin, and the secretion leader is Pbp or Azu.
8. The method of claim 5, wherein the host cell is defective in the expression of HslU and HslV, and the secretion leader is Pbp or Azu.
9. The method of claim 1, wherein the Pseudomonas host cell is wild- type and the
secretion leader is Pbp or Azu.
10. The method of claim 1, wherein the secretion leader is Azu, Pbp, IbpS31A, CupA2, or PbpA20V.
1 1. The method of claim 1, wherein said CRM197 nucleotide sequence has been
optimized for expression in the Pseudomonas host cell.
12. The method of claim 1, wherein the yield of soluble CRM197 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 1 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 1 1 g/L, about 12 g/L, about 0.5 g/L to about 1
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 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 1 1 g/L, about 0.5 g/L to about 12 g/L, about 1 g/L to about 2 g/L, about 1 g/L to about 3 g/L, about 1 g/L to about 4 g/L, about 1 g/L to about 5 g/L, about 1 g/L to about 6 g/L, about 1 g/L to about 7
g/L, about 1 g/L to about 8 g/L, about 1 g/L to about 9 g/L, about 1 g/L to about 10 g/L, about 1 g/L to about 1 1 g/L, about 1 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, 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 1 1 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 3 g/L to about 10 g/L, about 3 g/L to about 1 1 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 1 1 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 about 1 1 g/L, about 5 g/L to about 12 g/L,
3934938 l.DOC -32- WSGR Docket No. 38194-734.601 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 1 1 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 1 1 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 1 1 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 1 1 g/L, about 9 g/L to about 12 g/L, about 10 g/L to about 1 1 g/L, about 10 g/L to about 12 g/L, or about 1 1 g/L to about
1 g/L.
13. The method of claim 1, further comprising measuring the activity of the
recombinant CRM 197 protein in an activity assay, wherein about 40% to about 100% of the soluble
CRM 197 produced is determined to be active.
14. The method of claim 13, wherein the activity assay is an immunological assay or a receptor-binding assay.
15. The method of claim 1, wherein the expression vector comprises a lac derivative
promoter operatively linked to the protein coding sequence, and wherein the culturing comprises
induction of the 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.
16. The method of claim 1 , wherein the host cell is Pseudomonas fluoresceins.
3934938 l.DOC -33- WSGR Docket No. 38194-734.601
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