WO2011036455A1 - Souche bactérienne pour l'expression de protéines recombinantes, ayant une degp à activité protéase déficiente et conservant une activité chaperonne et des gènes tsp et ptr inactivés - Google Patents

Souche bactérienne pour l'expression de protéines recombinantes, ayant une degp à activité protéase déficiente et conservant une activité chaperonne et des gènes tsp et ptr inactivés Download PDF

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WO2011036455A1
WO2011036455A1 PCT/GB2010/001792 GB2010001792W WO2011036455A1 WO 2011036455 A1 WO2011036455 A1 WO 2011036455A1 GB 2010001792 W GB2010001792 W GB 2010001792W WO 2011036455 A1 WO2011036455 A1 WO 2011036455A1
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gene
mutated
protein
cell
degp
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PCT/GB2010/001792
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Mark Ellis
David Paul Humphreys
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Ucb Pharma S.A.
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Priority claimed from GB0916817A external-priority patent/GB0916817D0/en
Priority claimed from GB0916818A external-priority patent/GB0916818D0/en
Application filed by Ucb Pharma S.A. filed Critical Ucb Pharma S.A.
Publication of WO2011036455A1 publication Critical patent/WO2011036455A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, 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
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea

Definitions

  • the invention relates to a recombinant gram-negative bacterial host strain, particularly E. coli.
  • the invention also relates to a method for producing a protein of interest in such a recombinant bacterium.
  • Bacterial cells such as E. coli, are commonly used for producing recombinant proteins. There are many advantages to using bacterial cells, such as E. coli, for producing recombinant proteins particularly due to the versatile nature of bacterial cells as host cells allowing the gene insertion via plasmids. E. coli have been used to produce many recombinant proteins including human insulin.
  • protease sensitive proteins Despite the many advantages to using bacterial cells to produce recombinant proteins, there are still significant limitations including the difficulty of producing protease sensitive proteins. Proteases play an important role in turning over old and miss- folded proteins in the E. coli periplasm and cytoplasm. Bacterial proteases act to degrade the recombinant protein of interest, thereby often significantly reducing the yield of active protein.
  • proteases A number of bacterial proteases have been identified. In E. coli proteases including Protease III (ptr), DegP, OmpT, Tsp, prlC, ptrA, ptrB, pepA-T, tsh, espc, eatA, clpP and Ion have been identified.
  • Protease III ptr
  • DegP Protease III
  • OmpT OmpT
  • Tsp prlC
  • ptrA ptrA
  • ptrB pepA-T
  • tsh tsh
  • espc eatA
  • clpP and Ion Ion
  • the Protease HI (ptr) protein is a HOkDa periplasmic protease which degrades high molecular weight proteins.
  • Tsp (also known as Pre) is a 60kDa periplasmic protease.
  • the first known substrate of Tsp was Penicillin-binding protein-3 (PBP3) (Determination of the cleavage site involved in C-terminal processing of penicillin-binding protein 3 of Escherichia coli; Nagasawa H, Sakagami Y, Suzuki A, Suzuki H, Hara H, Hirota Y. J Bacteriol.
  • PBP3 Penicillin-binding protein-3
  • DegP also known as HtrA
  • HtrA is a 46kDa protein having dual function as a chaperone and a protease (Families of serine peptidases; Rawlings ND, Barrett AJ. Methods Enzymol. 1994;244: 19-61).
  • Meerman et al. (Construction and characterization of Escherichia coli strains deficient in All Known Loci Affecting the Proteolytic Stability of Secreted Recombinant Proteins. Meerman H. J., Georgeou G., Nature Biotechnology, 1994 Nov; 12;1 107- 1 1 10) disclose E. coli strains comprising mutations in the rpoH, the RNA polymerase sigma factor responsible for heat shock protein synthesis, and different combinations of mutations in protease genes including DegP, Protease III, Tsp(Prc) and OmpT, where null mutations of the protease genes were caused by insertional mutations.
  • US 5508192 discloses a method of producing recombinant polypeptides in protease-deficient bacterial hosts and constructs of single, double, triple and quadruple protease deficient bacteria which also carry a mutation in the rpoH gene.
  • Chen et al describes the construction of E. coli strains carrying different combinations of mutations in pre (Tsp) and DegP created by amplifying the upstream and downstream regions of the gene and ligating these together on a vector comprising selection markers and a sprW148R mutation (High-level accumulation of a recombinant antibody fragment in the periplasm of Escherichia coli requires a triple- mutant (ADegP Aprc sprW148R) host strain.
  • Chen C Snedecor B, Nishihara JC, Joly JC, McFarland N, Andersen DC, Battersby JE, Champion KM. Biotechnol Bioeng. 2004 Mar 5;85(5):463-74).
  • EP 1341899 discloses an E. coli strain that is deficient in chromosomal DegP and pre encoding proteases DegP and Pre, respectively, and harbors a mutant spr gene that encodes a protein that suppresses growth phenotypes exhibited by strains harboring pre mutants.
  • Kandilogiannaki et al (Expression of a recombinant human anti-MUCl scFv fragment in protease-deficient Escherichia coli mutants. Kandilogiannaki M, Koutsoudakis G, Zafiropoulos A, Krambovitis E. Int J Mol Med. 2001 Jun; 7(6): 659-64) describes the utilization of a protease deficient strain for the expression of a scFv protein.
  • protease-deficient strains have been used to improve the production recombinant proteins.
  • strains deficient in chromosomal DegP are generally temperature sensitive above about 28°C which means that such strains are not suitable for industrial purposes which typically require growth of cells at temperatures of 30°C to 37°C.
  • the present invention provides a recombinant gram-negative bacterial cell comprising: a. a mutated DegP gene encoding a DegP protein having chaperone activity and reduced protease activity; and b. a mutated Tsp gene, wherein the mutated Tsp gene encodes a Tsp protein having reduced protease activity or is a knockout mutated Tsp gene; and/or a c. a mutated ptr gene, wherein the mutated ptr gene encodes a Protease III protein having reduced protease activity or is a knockout mutated ptr gene .
  • the cell according to the present invention comprises a mutated DegP gene having chaperone activity and reduced protease activity.
  • the cell according to the present invention further comprises a mutated Tsp gene and/or a mutated ptr gene.
  • the present invention provides a recombinant gram-negative bacterial cell comprising a mutated DegP gene encoding a DegP protein having chaperone activity and reduced protease activity; and a mutated Tsp gene, wherein the mutated Tsp gene encodes a Tsp protein having reduced protease activity or is a knockout mutated Tsp gene.
  • the present invention provides a recombinant gram-negative bacterial cell comprising a mutated DegP gene encoding a DegP protein having chaperone activity and reduced protease activity, and a mutated ptr gene, wherein the mutated ptr gene encodes a Protease III protein having reduced protease activity or is a knockout mutated ptr gene.
  • the present invention provides a recombinant gram-negative bacterial cell comprising a mutated DegP gene encoding a DegP protein having chaperone activity and reduced protease activity, a mutated ptr gene, wherein the mutated ptr gene encodes a Protease III protein having reduced protease activity or is a knockout mutated ptr gene; and a mutated Tsp gene, wherein the mutated Tsp gene encodes a Tsp protein having reduced protease activity or is a knockout mutated Tsp gene.
  • the use of a mutated DegP gene encoding a DegP protein having chaperone activity and reduced protease activity in a gram-negative bacterium provides an advantageous host for producing recombinant protein of interest.
  • the cells provided by the present invention have reduced protease activity compared to a non-mutated cell, which may reduce proteolysis of a recombinant protein of interest, particularly proteins of interest which are proteolytically sensitive.
  • One or more of the gram-negative cells provided by the present invention may provide a high yield of a recombinant protein of interest.
  • One or more of the gram-negative cells provided by the present invention may provide a fast rate of production of a protein of interest.
  • One or more of the cells may provide fast initial yield of a recombinant protein of interest. Further, one or more of the cells may show good growth characteristics.
  • the present invention also provides a method for producing a recombinant protein of interest comprising expressing the recombinant protein of interest in a recombinant gram-negative bacterial cell comprising: a. a mutated DegP gene encoding a DegP protein having chaperone activity and reduced protease activity; and b. a mutated Tsp gene, wherein the mutated Tsp gene encodes a Tsp protein having reduced protease activity or is a knockout mutated Tsp gene; and/or a c. a mutated ptr gene, wherein the mutated ptr gene encodes a Protease III protein having reduced protease activity or is a knockout mutated ptr gene .
  • Figure 1 shows growth of E. coli strain MXE005 (carrying the DegP mutated gene and knockout mutated Tsp gene) compared to the wild type W31 10.
  • Figure 2 shows expression of Fab' in MXE005 compared to the wild type W31 10.
  • Figure 3 shows the expression of a Fab in MXE005 and W31 10.
  • Figure 4 shows the light chain (L chain), heavy chain (H chain) and Fab' expression during the course of a fermentation run in MXE005 and W31 10.
  • Figure 6 shows the growth profile of MXE005 compared to control W31 10.
  • Figure 7 shows Fab' yields from the supernatant (dotted lines) and periplasm (solid lines) of E. coli strain MXE005 and the W3110 control.
  • Figure 8 shows the total Fab' yield from the supernatant and periplasm of the E. coli strain MXE005 compared to W3110 control.
  • Figure 9 shows the Fab' specific production rate of E. coli strain MXE005 and the W31 10 control.
  • Figure 10a shows the 5' end of the wild type ptr (protease III) and knockout mutated ptr (protease III) protein and gene sequences.
  • Figure 10b shows the 5' end of the wild type Tsp and knockout mutated Tsp protein and gene sequences.
  • Figure 10c shows a region of the wild type DegP and mutated DegP protein and gene sequences.
  • SEQ ID NO: l is the DNA sequence of the non-mutated Tsp gene including the 6 nucleotides ATGAAC upstream of the start codon.
  • SEQ ID NO:2 is the amino acid sequence of the non-mutated Tsp protein.
  • SEQ ID NO:3 is the DNA sequence of a mutated knockout Tsp gene including the 6 nucleotides ATGAAT upstream of the start codon.
  • SEQ ID NO:4 is the DNA sequence of the non-mutated Protease III gene.
  • SEQ ID NO: 5 is the amino acid sequence of the non-mutated Protease III protein.
  • SEQ ID NO:6 is the DNA sequence of a mutated knockout Protease III gene.
  • SEQ ID NO:7 is the DNA sequence of the non-mutated DegP gene.
  • SEQ ID NO:8 is the amino acid sequence of the non-mutated DegP protein.
  • SEQ ID NO:9 is the DNA sequence of a mutated DegP gene.
  • SEQ ID NO: 10 is the amino acid sequence of a mutated DegP protein.
  • SEQ ID NO: 1 1 is the amino acid sequence of the light chain variable region of an anti-TNF antibody.
  • SEQ ID NO: 12 is the amino acid sequence of the heavy chain variable region of an anti-TNF antibody.
  • SEQ ID NO: 13 is the amino acid sequence of the light chain of an anti-TNF antibody.
  • SEQ ID NO: 14 is the amino acid sequence of the heavy chain of an anti-TNF antibody.
  • SEQ ID NO: 15 is the sequence of the 3' oligonucleotide primer for the region of the mutated Tsp gene comprising the Ase I restriction site.
  • SEQ ID NO: 16 is the sequence of the 5' oligonucleotide primer for the region of the mutated Tsp gene comprising the Ase I restriction site.
  • SEQ ID NO: 17 is the sequence of the 3' oligonucleotide primer for the region of the mutated Protease III gene comprising the Ase I restriction site.
  • SEQ ID NO: 18 is the sequence of the 5' oligonucleotide primer for the region of the mutated Protease III gene comprising the Ase I restriction site.
  • SEQ ID NO: 19 is the sequence of the 5' oligonucleotide primer for the region of the mutated DegP gene comprising the Ase I restriction site.
  • SEQ ID NO: 20 is the sequence of the 3' oligonucleotide primer for the region of the mutated DegP gene comprising the Ase I restriction site.
  • one or more recombinant gram-negative bacterial cells provided by the present invention has advantageous characteristics for producing a protein of interest due to modification of the cell's genome to mutate the DegP gene so that it encodes a DegP protein having chaperone activity and reduced protease activity and introduce mutations to ptr and/or Tsp which encode proteins having reduced protease activity or which are knockout mutations.
  • the cells provided by the present invention have reduced protease activity compared to non-mutated cell, which may reduce proteolysis of a recombinant protein of interest, particularly proteins of interest which are proteolytically sensitive.
  • one or more of the gram-negative cells provided by the present invention may provide higher yield of the intact recombinant protein of interest and a lower yield, or preferably no yield, of proteolytic fragments of the protein of interest compared to a non-mutated bacterial cell.
  • One or more of the recombinant bacterial cells of the present invention may exhibit significantly improved protein yield compared to a non-mutated bacterial cell.
  • the improved protein yield may be the periplasmic protein yield and/or the supernatant protein yield.
  • One or more of the recombinant bacterial cells of the present invention may be capable of faster rate of production of a protein of interest and, therefore, the same quantity of a protein of interest may be produced in a shorter time compared to a non-mutated bacterial cell.
  • the faster rate of production of a protein of interest may be especially significant over the initial period of growth of the cell, for example over the first 5, 10, 20 or 30 hours post induction of protein expression.
  • one or more of the cells according to the present invention may exhibit improved protein yield compared to a cell only carrying the DegP mutation and/or a cell carrying the ptr mutation or the Tsp mutation.
  • the cell according to the present invention comprises the combination of the DegP mutation and the Tsp mutation.
  • a cell comprising the combination of the DegP mutation and the Tsp knockout mutation is particularly preferred.
  • This cell exhibits a significantly high yield and a fast initial yield of a protein of interest compared to a non-mutated cell.
  • a specific example of such a cell line comprising the mutated DegP gene and a mutated Tsp gene is mutant E. coli cell strain MXE005 having genotype ATsp, DegP S210A and deposited on 21 st May 2009 at the National Collection of Type Cultures, HPA, United Kingdom, under Accession number NCTC13448.
  • one or more of the cells provided by the present invention may provide improved yield of correctly folded proteins from the cell relative to non- mutated cells or to mutated cells wherein the DegP gene has been mutated to knockout DegP thereby preventing DegP expression, such as chromosomal deficient DegP.
  • the chaperone activity of DegP is lost completely whereas in the cell according to the present invention the chaperone activity of DegP is retained whilst the protease activity is lost.
  • the one or more cells according to the present invention have a lower protease activity to prevent proteolysis of the protein whilst maintaining the chaperone activity to allow correct folding and transportation of the protein in the host cell.
  • one or more of the cells may show good growth characteristics including cell growth and/or reproduction which may be substantially the same as a non-mutated bacterial cell or improved compared to a non-mutated bacterial cell.
  • one or more of the cells may have improved cell growth and/or reproduction compared to a cell only carrying the DegP mutation and/or a cell carrying the ptr mutation or the Tsp mutation
  • One or more cells according to the present invention may have improved cell growth compared to cells carrying a mutated knockout DegP gene preventing DegP expression.
  • improved cell growth maybe exhibited due to the DegP protease retaining chaperone activity which may increase capacity of the cell to process all proteins which require chaperone activity. Accordingly, the production of correctly folded proteins necessary for the cell's growth and reproduction may be increased in one or more of the cells of the present invention compared to cells carrying a DegP knockout mutation preventing DegP expression thereby improving the cellular pathways regulating growth.
  • known DegP protease deficient strains are generally temperature-sensitive and do not typically grow at temperatures higher than about 28°C. However, the cells according to the present invention are not temperature-sensitive and may be grown at temperatures of 28°C or higher, including temperatures of approximately 30°C to approximately 37°C, which are typically used for industrial scale production of proteins from bacteria.
  • Suitable methods to measure the expression of a protein of interest and growth characteristics of a cell are well known in the art including fermentation methods, ELISA and protein G hplc. Suitable fermentation methods are described in Humphreys D P, et al. (1997). Formation of dimeric Fabs in E. coli: effect of hinge size and isotype, presence of interchain disulphide bond, Fab' expression levels, tail piece sequences and growth conditions. J. IMMUNOL. METH. 209: 193-202; Backlund E. Reeks D. Markland K. Weir N. Bowering L. Larsson G. Fedbatch design for periplasmic product retention in Escherichia coli, Journal Article. Research Support, Non-U.S.
  • protein and “polypeptide” are used interchangeably herein, unless the context indicates otherwise.
  • Peptide is intended to refer to 10 or less amino acids.
  • polynucleotide includes a gene, DNA, cDNA, RNA, mR A etc unless the context indicates otherwise.
  • the non-mutated cell or control cell in the context of the present invention means a cell of the same type as the recombinant gram-negative cell of the invention wherein the cell has not been modified to carry mutated DegP gene encoding a DegP protein having chaperone activity but not protease activity; and a mutated ptr gene encoding Protease III and/or a mutated Tsp gene encoding protease Tsp.
  • a non- mutated cell may be a wild-type cell and may be derived from the same population of host cells as the cells of the invention before modification to introduce the mutations.
  • DegP means a gene encoding DegP protein (also known as HtrA), which has dual function as a chaperone and a protease (Families of serine peptidases; Rawlings ND, Barrett AJ. Methods Enzymol. 1994;244:19-61).
  • the sequence of the non-mutated DegP gene is shown in SEQ ID NO: 7 and the sequence of the non- mutated DegP protein is shown in SEQ ID NO: 8.
  • DegP functions as a chaperone and at high temperatures DegP has a preference to function as a protease (A Temperature-Dependent Switch from Chaperone to Protease in a Widely conserveed Heat Shock Protein. Cell, Volume 97 , Issue 3 , Pages 339 - 347. Spiess C, Beil A, Ehrmann M and The proteolytic activity of the HtrA (DegP) protein from Escherichia coli at low temperatures, Skorko-Glonek J et al Microbiology 2008, 154, 3649-3658).
  • protease A Temperature-Dependent Switch from Chaperone to Protease in a Widely conserveed Heat Shock Protein. Cell, Volume 97 , Issue 3 , Pages 339 - 347. Spiess C, Beil A, Ehrmann M and The proteolytic activity of the HtrA (DegP) protein from Escherichia coli at low temperatures, Skorko-Gl
  • the DegP mutation in the cell of the present invention provides a mutated DegP gene encoding a DegP protein having chaperone activity and reduced protease activity.
  • the expression "having chaperone activity" in the context of the present invention means that the mutated DegP protein has the same or substantially the same chaperone activity compared to the wild-type non-mutated DegP protein.
  • the mutated DegP gene encodes a DegP protein having 50% or more, 60% or more, 70% or more, 80% or more, 90% or more or 95% or more of the chaperone activity of a wild-type non-mutated DegP protein. More preferably, the mutated DegP gene encodes a DegP protein having the same chaperone activity compared to wild-type DegP.
  • the expression "having reduced protease activity" in the context of the present invention means that the mutated DegP protein does not have the full protease activity compared to the wild-type non-mutated DegP protein.
  • the mutated DegP gene encodes a DegP protein having 50% or less, 40% or less, 30% or less, 20% or less, 10% or less or 5% or less of the protease activity of a wild-type non-mutated DegP protein. More preferably, the mutated DegP gene encodes a DegP protein having no protease activity.
  • the cell is not deficient in chromosomal DegP i.e. the DegP gene sequence has not been deleted or mutated to prevent expression of any form of DegP protein.
  • Any suitable mutation may be introduced into the DegP gene in order to produce a protein having chaperone activity and reduced protease activity.
  • the protease and chaperone activity of a DegP protein expressed from a gram-negative bacterium may be easily tested by a person skilled in the art by any suitable method such as the method described in Spiess et al wherein the protease and chaperone activities of DegP were tested on MalS, a natural substrate of DegP (A Temperature-Dependent Switch from Chaperone to Protease in a Widely conserveed Heat Shock Protein. Cell, Volume 97 , Issue 3 , Pages 339 - 347.
  • DegP HtrA
  • the DegP mutation to produce a protein having chaperone activity and reduced protease activity may comprise a mutation, such as a missense mutation to one, two or three of Hisl05, Aspl35 and Ser210.
  • the mutated DegP gene may comprise:
  • One, two or three of His 105, Asp 135 and Ser210 may be mutated to any suitable amino acid which results in a protein having chaperone activity and reduced protease activity.
  • one, two or three of Hisl05, Aspl 35 and Ser210 may be mutated to a small amino acid such as Gly or Ala.
  • a further suitable mutation is to change one, two or three of Hisl05, Aspl 35 and Ser210 to an amino acid having opposite properties such as Asp 135 being mutated to Lys or Arg, polar His 105 being mutated to a non-polar amino acid such as Gly, Ala, Val or Leu and small hydrophilic Ser210 being mutated to a large or hydrophobic residue such as Val, Leu, Phe or Tyr.
  • the DegP gene comprises the point mutation S210A, as shown in Figure 10c, which has been found to produce a protein having chaperone activity but not protease activity (A Temperature-Dependent Switch from Chaperone to Protease in a Widely conserveed Heat Shock Protein. Cell, Volume 97 , Issue 3 , Pages 339 - 347. Spiess C, Beil A, Ehrmann M).
  • the present invention also provides a recombinant gram-negative bacterial cell comprising a mutated DegP gene encoding a DegP protein having chaperone activity and reduced protease activity, wherein the DegP gene comprises a mutation to His 105; or a mutation to Asp 135; or a mutation to His 105 and Asp 135; or a mutation to Hisl05 and Ser210; or a mutation to Aspl35 and Ser210; or a mutation to Hisl05, Aspl35 and Ser210, as discussed above.
  • DegP has two PDZ domains, PDZ1 (residues 260-358) and PDZ2 (residues 359-448), which mediate protein-protein interaction (A Temperature-Dependent Switch from Chaperone to Protease in a Widely conserveed Heat Shock Protein. Cell, Volume 97 , Issue 3 , Pages 339 - 347. Spiess C, Beil A, Ehrmann M).
  • the degP gene is mutated to delete PDZ1 domain and/or PDZ2 domain.
  • PDZ 1 and PDZ2 results in complete loss of protease activity of the DegP protein and lowered chaperone activity compared to wild-type DegP protein whilst deletion of either PDZ1 or PDZ2 results in 5% protease activity and similar chaperone activity compared to wild-type DegP protein (A Temperature- Dependent Switch from Chaperone to Protease in a Widely conserveed Heat Shock Protein. Cell, Volume 97 , Issue 3 , Pages 339 - 347. Spiess C, Beil A, Ehrmann M).
  • the present invention also provides a recombinant gram-negative bacterial cell comprising a mutated DegP gene encoding a DegP protein having chaperone activity and reduced protease activity, wherein the degP gene is mutated to delete PDZ1 domain and/or PDZ2 domain, as discussed above.
  • the mutated DegP gene may also comprise a silent non-naturally occurring restriction site, such as Ase I in order to aid in identification and screening methods, for example as shown in Figure 10c.
  • the preferred sequence of the mutated DegP gene comprising the point mutation S210A and an Ase I restriction marker site is provided in SEQ ID NO: 9 and the encoded protein sequence is shown in SEQ ID NO: 10.
  • the mutations which have been made in the mutated DegP sequence of SEQ ID NO: 9 are shown in Figure 10c.
  • the present invention also provides a recombinant gram-negative bacterial cell comprising a mutated DegP gene encoding a DegP protein having chaperone activity and reduced protease activity; and a polynucleotide sequence encoding a heterologous protein of interest.
  • ptr gene means a gene encoding Protease III, a protease which degrades high molecular weight proteins.
  • SEQ ID NO: 4 The sequence of the non-mutated ptr gene is shown in SEQ ID NO: 4 and the sequence of the non-mutated Protease III protein is shown in SEQ ID NO: 5.
  • Tsp gene means a gene encoding protease Tsp (also known as Pre) which is a periplasmic protease capable of acting on Penicillin-binding protein-3 (PBP3) and phage tail proteins.
  • PBP3 Penicillin-binding protein-3
  • SEQ ID NO: 1 The sequence of the non-mutated Tsp gene is show in SEQ ID NO: 1 and the sequence of the non- mutated Tsp protein is shown in SEQ ID NO: 2.
  • Reference to the mutated ptr gene or mutated ptr gene encoding Protease III refers to either a mutated ptr gene encoding a Protease III protein having reduced protease activity or a knockout mutated ptr gene, unless otherwise indicated.
  • mutated Tsp gene or mutated Tsp gene encoding Tsp refers to either a mutated Tsp gene encoding a Tsp protein having reduced protease activity or a knockout mutated Tsp gene, unless otherwise indicated.
  • mutated ptr gene encoding a Protease III protein having reduced protease activity and “mutated Tsp gene encoding a Tsp protein having reduced protease activity” in the context of the present invention means that the mutated ptr gene or the mutated Tsp gene does not have the full protease activity compared to the wild-type non-mutated ptr gene or Tsp gene.
  • the mutated ptr gene encodes a Protease III having 50% or less, 40% or less, 30% or less, 20% or less, 10% or less or 5% or less of the protease activity of a wild-type non-mutated Protease III protein. More preferably, the mutated ptr gene encodes a Protease III protein having no protease activity.
  • the cell is not deficient in chromosomal ptr i.e. the ptr gene sequence has not been deleted or mutated to prevent expression of any form of Protease III protein. Any suitable mutation may be introduced into the ptr gene in order to produce a Protease III protein having reduced protease activity.
  • the protease activity of a Protease III protein expressed from a gram-negative bacterium may be easily tested by a person skilled in the art by any suitable method in the art.
  • the mutated Tsp gene encodes a Tsp protein having 50% or less, 40% or less, 30% or less, 20% or less, 10% or less or 5% or less of the protease activity of a wild-type non-mutated Tsp protein. More preferably, the mutated Tsp gene encodes a Tsp protein having no protease activity. In this embodiment the cell is not deficient in chromosomal Tsp i.e. the Tsp gene sequence has not been deleted or mutated to prevent expression of any form of Tsp protein.
  • Any suitable mutation may be introduced into the Tsp gene in order to produce a protein having reduced protease activity.
  • the protease activity of a Tsp protein expressed from a gram-negative bacterium may be easily tested by a person skilled in the art by any suitable method in the art, such as the method described in Keiler et al (Identification of Active Site Residues of the Tsp Protease* THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 270, No. 48, Issue of December 1, pp. 28864- 28868, 1995 Kenneth C. Keiler and Robert T. Sauer) wherein the protease activities of Tsp was tested.
  • Tsp has been repored in Keiler et al ⁇ supra) as having an active site comprising residues S430, D441 and K455 and residues G375, G376, E433 and T452 are important for maintaining the structure of Tsp.
  • Keiler et al ⁇ supra reports findings that the mutated Tsp genes S430A, D441A, K455A, K455H, K455R, G375A, G376A, E433A and T452A had no detectable protease activity. It is further reported that the mutated Tsp gene S430C displayed about 5-10% wild-type activity.
  • the Tsp mutation to produce a protein having reduced protease activity may comprise a mutation, such as a missense mutation to one or more of residues S430, D441 , K455, G375, G376, E433 and T452.
  • the Tsp mutation to produce a protein having reduced protease activity may comprise a mutation, such as a missense mutation to one, two or all three of the active site residues S430, D441 and K455.
  • the mutated Tsp gene may comprise:
  • S430, D441, 455, G375, G376, E433 and T452 may be mutated to any suitable amino acid which results in a protein having reduced protease activity.
  • suitable mutations are S430A, S430C, D441A, K455A, 455H, K455R, G375A, G376A, E433A and T452A.
  • the mutated Tsp gene may comprise one, two or three mutations to the active site residues, for example the gene may comprise:
  • the Tsp gene comprises the point mutation S430A or S430C.
  • the present invention also provides a recombinant gram-negative bacterial cell comprising a mutated Tsp gene, wherein the mutated Tsp gene encodes a Tsp protein having reduced protease activity, wherein the Tsp gene comprise a mutation, such as a missense mutation to one or more of residues S430, D441, K455, G375, G376, E433 and T452, as discussed above.
  • knockout mutated ptr gene and “knockout mutated Tsp gene” in the context of the present invention means that the gene comprises one or more mutations thereby causing no expression of the protein encoded by the gene to provide a cell deficient in the protein encoded by the knockout mutated gene.
  • the knockout gene may be partially or completely transcribed but not translated into the encoded protein.
  • the ptr gene and/or Tsp gene may be mutated in any suitable way, for example by one or more deletion, insertion, point, missense, nonsense and frameshift mutations, to cause no expression of the protein.
  • the gene may be knocked out by insertion of a foreign DNA sequence, such as an antibiotic resistance marker, into the gene coding sequence.
  • Knockout mutations to protease encoding genes have been routinely carried out previously by insertion of a DNA sequence into the gene coding sequence.
  • the inserted DNA sequence typically codes for a selection marker such as an antibiotic resistance gene.
  • this mutation method may be effective at knocking out the target protease, there are many disadvantages associated with this method.
  • One disadvantage is the insertion of the foreign DNA, such as an antibiotic resistance gene, causes disruption in the host's genome which may result in any number of unwanted effects including the over-expression of other proteins and/or down- regulation or knockout of other proteins. This effect is particularly evident for those genes positioned immediately upstream or downstream of the target protease gene.
  • a further disadvantage to the insertion of foreign DNA, particularly antibiotic resistance genes is the unknown phenotypic modifications to the host cell which may affect expression of the target protein and/or growth of the host cell and may also make the host cell unsuitable for production of proteins intended for use as therapeutics.
  • Antibiotic resistance proteins are particularly disadvantageous for biosafety requirements of large scale manufacturing particularly for the production of therapeutics for human administration.
  • a further disadvantage to the insertion of antibiotic resistance markers is the metabolic burden on the cell created by the expression of the protein encoded by the antibiotic resistance gene. The use of antibiotic resistance markers for use as markers for genetic manipulations such as knockout mutations, are also limited by the number of different antibiotic resistance markers available.
  • the gene is not mutated by insertion of a foreign DNA sequence, such as an antibiotic resistance marker, into the gene coding sequence.
  • a foreign DNA sequence such as an antibiotic resistance marker
  • the above disadvantages of known knockout mutation methods are overcome by creating knockout mutations to the ptr gene and/or the Tsp gene by a missense mutation to the gene start codon and or one or more stop codons positioned downstream of the gene start codon and upstream of the gene stop codon.
  • a missense mutation to the target knockout gene start codon ensures that the target gene does not comprise a suitable start codon at the start of the coding sequence.
  • the missense mutation to the start codon may be a mutation of one, two or all three of the nucleotides of the start codon. Alternatively or additionally the start codon may be mutated by an insertion or deletion frameshift mutation.
  • the ptr gene and Tsp gene each comprise an ATG start codon. If the gene comprises more than one suitably positioned start codon, as found in the Tsp gene where two ATG codons are present at the 5' end of the coding sequence, one or both of the ATG codons may be mutated by a missense mutation.
  • the ptr gene is mutated to change the ATG start codon to ATT, as shown in Figure 10a.
  • the Tsp gene is mutated at the second ATG codon (codon 3) to TCG, as shown in Figure 10b.
  • the knockout mutated ptr gene and/or the knockout mutated Tsp gene may alternatively or additionally comprise one or more stop codons positioned downstream of the gene start codon and upstream of the gene stop codon.
  • the knockout mutated ptr gene and/or the knockout mutated Tsp gene comprise both a missense mutation to the start codon and one or more inserted stop codons.
  • the one or more inserted stop codons are preferably in-frame stop codons. However one or more inserted stop codons may alternatively or additionally be out-of-frame stop codons. One or more out-of-frame stop codons may be required to stop translation where an out-of-frame start codon is changed to an in-frame start codon by an insertion or deletion frameshift mutation.
  • the one or more stop codons may be introduced by any suitable mutation including a nonsense point mutation and a frameshift mutation.
  • the one or more stop codons are preferably introduced by a frameshift mutation and/or an insertion mutation, preferably by replacement of a segment of the gene sequence with a sequence comprising a stop codon. For example an Ase I restriction site may be inserted, which comprises the stop codon TAA.
  • the ptr gene is mutated to insert an in-frame stop codon by insertion of an Ase I restriction site, as shown in Figure 10a.
  • the Tsp gene is mutated to delete "T" from the fifth codon thereby causing a frameshift resulting in stop codons at codons 1 1 and 16, as shown in Figure 10b.
  • the Tsp gene is mutated to insert an Ase I restriction site to create a third in-frame stop codon at codon 21 , as shown in Figure 10b.
  • the knockout mutated ptr gene has the DNA sequence of SEQ ID NO: 6.
  • the mutations which have been made in the knockout mutated ptr gene sequence of SEQ ID NO: 6 are shown in Figure 10a.
  • the knockout mutated Tsp gene has the DNA sequence of SEQ ID NO: 3, which includes the 6 nucleotides ATGAAT upstream of the start codon.
  • the mutations which have been made in the knockout mutated Tsp sequence of SEQ ID NO: 3 are shown in Figure 10b.
  • the mutated Tsp gene has the DNA sequence of nucleotides 7 to 2048 of SEQ ID NO:3.
  • knockout mutations are advantageous because they cause minimal or no disruption to the chromosomal DNA upstream or downstream of the target knockout gene site and do not require the insertion and retention of foreign DNA, such as antibiotic resistance markers, which may affect the cell's suitability for expressing a protein of interest, particularly therapeutic proteins. Accordingly, one or more of the cells according to the present invention may exhibit improved growth characteristics and/or protein expression compared to cells wherein the protease gene has been knocked out by insertion of foreign DNA.
  • a further embodiment of the present invention overcomes the above disadvantages of using antibiotic resistance markers wherein the mutated DegP gene, the mutated ptr gene and/or the mutated Tsp gene are mutated to comprise one or more restriction marker sites.
  • the restriction sites are genetically engineered into the gene and are non-naturally occurring.
  • the restriction marker sites are advantageous because they allow screening and identification of correctly modified cells which comprise the required chromosomal mutations.
  • Cells which have been modified to carry one or more of the mutated protease genes may be analyzed by PCR of genomic DNA from cell lysates using oligonucleotide pairs designed to amplify a region of the genomic DNA comprising a non-naturally occurring restriction marker site.
  • the amplified DNA may then be analyzed by agarose gel electrophoresis before and after incubation with a suitable restriction enzyme capable of digesting the DNA at the non-naturally occurring restriction marker site.
  • a suitable restriction enzyme capable of digesting the DNA at the non-naturally occurring restriction marker site.
  • the presence of DNA fragments after incubation with the restriction enzyme confirms that the cells have been successfully modified to carry the one or more mutated protease genes.
  • the oligonucleotide primer sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18 may be used to amplify the region of the DNA comprising the non- naturally occurring Ase I restriction site from the genomic DNA of transformed cells.
  • the amplified genomic DNA may then be incubated with Ase I restriction enzyme and analyzed by gel electrophoresis to confirm the presence of the mutated ptr gene in the genomic DNA.
  • the knockout mutated Tsp gene has the DNA sequence of SEQ ID NO: 3 or nucleotides 7 to 2048 of SEQ ID NO:3, the oligonucleotide primer sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16 may be used to amplify the region of the DNA comprising the non-naturally occurring Ase I restriction site from the genomic DNA of transformed cells.
  • the amplified genomic DNA may then be incubated with Ase I restriction enzyme and analyzed by gel electrophoresis to confirm the presence of the mutated Tsp gene in the genomic DNA.
  • the oligonucleotide primer sequences shown in SEQ ID NO: 19 and SEQ ID NO:20 may be used to amplify the region of the DNA comprising the non-naturally occurring Ase I restriction site from the genomic DNA of transformed cells.
  • the amplified genomic DNA may then be incubated with Ase I restriction enzyme and analyzed by gel electrophoresis to confirm the presence of the mutated DegP gene in the genomic DNA.
  • the one or more restriction sites may be introduced by any suitable mutation including by one or more deletion, insertion, point, missense, nonsense and frameshift mutations.
  • a restriction site may be introduced by the mutation of the start codon and/or mutation to introduce the one or more stop codons, as described above. This embodiment is advantageous because the restriction marker site is a direct and unique marker of the knockout mutations introduced.
  • a restriction maker site may be inserted which comprises an in-frame stop codon, such as an Ase I restriction site.
  • an in-frame stop codon such as an Ase I restriction site.
  • the inserted restriction site serves as both a restriction marker site and a stop codon to prevent full transcription of the gene coding sequence.
  • a stop codon is introduced to the ptr gene by introduction of an Ase I site
  • this also creates a restriction site, as shown in Figure 10a.
  • a stop codon is introduced to the Tsp gene at codon 21 by introduction of an Ase I site, this also creates a restriction site, as shown in Figure 10b.
  • a restriction marker site may be inserted by the missense mutation to the start codon and optionally one or more further point mutations.
  • the restriction marker site is preferably an EcoR 1 restriction site. This is particularly advantageous because the missense mutation to the start codon also creates a restriction marker site. For example, in the embodiment wherein the start codon of the ptr gene is changed to ATT, this creates an EcoR I marker site, as shown in Figure 10a.
  • a marker restriction site may be introduced using silent codon changes.
  • an Ase I site may be used as a silent restriction marker site, wherein the TAA stop codon is out-of-frame, as shown in Figure 10c.
  • one or more marker restriction site may be introduced using silent codon changes.
  • the recombinant gram-negative bacterial cell according to the present invention may be produced by any suitable means.
  • suitable techniques which may be used to replace a chromosomal gene sequence with a mutated gene sequence.
  • Suitable vectors may be employed which allow integration into the host chromosome by homologous recombination. Suitable gene replacement methods are described, for example, in Hamilton et al (New Method for Generating Deletions and Gene Replacements in Escherichia coli, Hamilton C. M. et al , Journal of Bacteriology Sept. 1989, Vol. 171, No. 9 p 4617-4622), Skorupski et al (Positive selection vectors for allelic exchange, Skorupski K and Taylor R.
  • Kiel et al A general method for the construction of Escherichia coli mutants by homologous recombination and plasmid segregation, Kiel J.A.K.W. et al, Mol Gen Genet 1987, 207:294-301), Blomfield et al (Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature sensitive pSCl Ol replicon, Blomfield I. C. et al , Molecular Microbiology 1991, 5(6), 1447-1457) and Ried et al.
  • nptl-sacB-sacR cartridge for constructing directed, unmarked mutations in Gram-negative bacteria by marker exchange-eviction mutagenesis, Ried J. L. and Collmer A., Gene 57 (1987) 239-246).
  • a suitable plasmid which enables homologous recombination/replacement is the pK03 plasmid (Link et al., 1997, Journal of Bacteriology, 179, 6228-6237).
  • Successfully mutated strains may be identified using methods well known in the art including colony PCR DNA sequencing and colony PCR restriction enzyme mapping.
  • the present invention provides a mutant E. coli cell strain MXE005 having genotype ATsp, DegP S210A and deposited on 21 st May 2009 at the National Collection of Type Cultures, Health Protection Agency (HP A), Centre for Infections, 61 Colindale Avenue, London, NW9 5EQ, United Kingdom, under Accession number NCTC 13448.
  • the present invention provides a mutant E. coli cell strain MXE006 having genotype Aptr, DegP S21 OA and deposited on 21 st May 2009 at the National Collection of Type Cultures, Health Protection Agency (HP A), Centre for Infections, 61 Colindale Avenue, London, NW9 5EQ, United Kingdom, under Accession number NCTC 13449.
  • the mutated DegP gene and mutated ptr and/or Tsp genes may be introduced into the gram-negative bacterium on the same or different vectors.
  • the present invention also provides a vector capable of integration into a bacterial chromosome, wherein the vector comprising a mutated DegP gene encoding a DegP protein having chaperone activity but not protease activity; and a mutated ptr gene encoding Protease III and/or mutated Tsp encoding protease Tsp.
  • the gram-negative bacterial cell according to the present invention does not carry a mutated ompT gene, such as being deficient in chromosomal ompT. In one embodiment the cell according to the present invention does not carry any further mutated protease genes apart from the mutated ptr gene and/or the mutated Tsp gene.
  • Any suitable gram-negative bacterium may be used as the parental cell for producing the recombinant cell of the present invention.
  • Suitable gram-negative bacterium include Salmonella typhimurium, Pseudomonas fluorescens, Erwinia carotovora, Shigella, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, Acinetobacter baumannii and E. coli.
  • the parental cell is E. coli. Any suitable strain of E. coli may be used in the present invention as the parental cell. Examples of suitable E.
  • coli strains include the K.-12 strain family which comprises W31 10 (F ⁇ " rph-1 INV(rrnD, rrnE) ilvG) (ATCC27325), MG 1655 (F ⁇ ' ilvG- rfb-50 rph-1) (ATCC700926), W1485 (F+ ⁇ " rph-1 rpoS396) (ATCC12435), W3101 (F ⁇ " ilvG- IN(rrnD-rrnE)l rph-1 galT22) and BW30270 (F ⁇ " fnr+).
  • E. coli strains include the W strain (ATCC9637) and the B strain (ATCC23226).
  • a wild-type W31 10 strain such as K-12 W31 10, is used.
  • strains of bacteria are known and used for expressing recombinant proteins.
  • mutated bacteria such as E. coli
  • strains are used for expressing recombinant proteins wherein genes involved in cell metabolism and DNA replication are mutated such as, for example phoA, fhuA, lac, rec, gal,ara, arg, thi and pro.
  • genes involved in cell metabolism and DNA replication are mutated such as, for example phoA, fhuA, lac, rec, gal,ara, arg, thi and pro.
  • These mutations may have many deleterious effects on the host cell including effects on cell growth, stability, recombinant protein expression yield and toxicity.
  • Strains having one or more of these genomic mutations, particularly strains having a high number of these mutations may exhibit a loss of fitness which reduces bacterial growth rate to a level which is not suitable for industrial protein production. Further, any of the above genomic mutations may affect other genes in cis and/or in trans in unpredictable harmful ways thereby alter
  • the cell according to the present invention is isogenic to a wild-type bacterial cell except for the mutated DegP gene, the mutated ptr gene and/or the mutated Tsp gene and optionally a polynucleotide encoding a protein of interest.
  • This embodiment is particularly advantageous because the cell carries only minimal mutations to the genomic sequence in order to introduce the above protease mutations and does not carry any other mutations which may have deleterious effects on the cell's growth and ability to express a protein of interest. Accordingly, the cell according to the present invention may exhibit improved growth characteristics and/or protein expression compared to cells comprising further genetically engineered mutations to the genomic sequence. The cell is also more suitable for producing therapeutic proteins.
  • the present invention provides a cell which is isogenic to a wild-type bacterial cell except for the mutated DegP gene and the mutated Tsp gene and optionally a polynucleotide sequence encoding a protein of interest.
  • the present invention also provides a cell which is isogenic to a wild-type bacterial cell except for the mutated DegP gene and mutated ptr gene and optionally a polynucleotide sequence encoding a protein of interest.
  • the cell according to the present invention is isogenic to a wild-type E. coli cell except for the above protease mutations and optionally the polynucleotide encoding a protein of interest. More preferably the cell according to the present invention is isogenic to an E. coli strain W3110 except for the above protease mutations and optionally the polynucleotide encoding a protein of interest. Examples of other suitable wild-type E.
  • coli cells which the cell according to the present invention may be isogenic to except for the above protease mutations and optionally the polynucleotide encoding a protein of interest are strains of the K- 12 strain family which includes W31 10 (F ⁇ " rph-1 INV(rrnD, rrnE) ilvG) (ATCC27325), MG1655 (F ⁇ ' ilvG- rfb-50 ⁇ - ⁇ ) (ATCC700926), W1485 (F+ ⁇ " ⁇ -l rpoS396) (ATCC12435), W3101 (F ⁇ " ilvG- iN(rrnD-rrnE) 1 ⁇ - ⁇ galT22) and BW30270 (F ⁇ " fnr+).
  • E. coli strains which the cell according to the present invention may be isogenic to except for the above protease mutations and optionally the polynucleotide encoding a protein of interest are include the W strain (ATCC9637) and the B strain (ATCC23226).
  • the term "isogenic" in the context of the present invention means that the genome of the cell of the present invention has substantially the same or the same genomic sequence compared to wild-type cell except for the mutated DegP gene, the mutated ptr and/or the mutated Tsp gene and optionally a polynucleotide encoding a protein of interest.
  • the cell according to the present invention comprises no further non-naturally occurring or genetically engineered mutations compared to the wild-type cell.
  • the cell according to the present invention may have substantially the same genomic sequence compared to the wild-type cell except for the above protease mutations and optionally a polynucleotide encoding a protein of interest taking into account any naturally occurring mutations which may occur.
  • the cell according to the present invention may have substantially the same genomic sequence compared to the wild-type cell except for the above protease mutations and optionally a polynucleotide encoding a protein of interest taking into account any naturally occurring mutations which may occur and any further genomic mutations which may result from the introduction of the protease mutations and/or the polynucleotide encoding the protein of interest. Examples of gene mutations involved in cell metabolism and DNA replication, which are commonly used in E.
  • the cell according to the present invention may have exactly the same genomic sequence compared to the wild- type cell except for the above protease mutations and optionally a polynucleotide encoding a protein of interest.
  • the cell of the present invention may further differ from a wild-type cell by comprising a polynucleotide encoding the protein of interest.
  • the polynucleotide encoding the protein of interest may be contained within a suitable expression vector transformed into the cell and/or integrated into the host cell's genome.
  • the cell of the present invention will also differ from a wild-type cell due to the inserted polynucleotide sequence encoding the protein of interest.
  • the polynucleotide is in an expression vector in the cell thereby causing minimal disruption to the host cell's genome.
  • wild-type in the context of the present invention means a strain of a gram- negative bacterial cell as it may occur in nature or may be isolated from the environment, which does not carry any genetically engineered mutations.
  • An example of a wild-type strain of E. colt is W31 10, such as W31 10 K-12 strain.
  • wild-type strains include strains of the K-12 strain family which includes W31 10 (F “ ⁇ " rph-1 I V(rrnD, rrnE) ilvG) (ATCC27325), MG1655 (F ⁇ " ilvG- rfb-50 rph-1) (ATCC700926), W1485 (F+ ⁇ ' rph-1 rpoS396) (ATCC 12435), W3101 (F ⁇ " ilvG- IN(rrnD-rrnE)l rph-1 galT22) and BW30270 (F ⁇ ' fnr+).
  • Further examples of wild-type E. coli strains include the W strain (ATCC9637) and the B strain (ATCC23226).
  • the cell according to the present invention may further comprise a polynucleotide sequence encoding a protein of interest.
  • the polynucleotide sequence encoding the protein of interest may be exogenous or endogenous.
  • the polynucleotide sequence encoding the protein of interest may be integrated into the host's chromosome or may be non-integrated in a vector, typically a plasmid.
  • the cell according to the present invention expresses a protein of interest.
  • Protein of interest in the context of the present specification is intended to refer to polypeptide for expression, usually a recombinant polypeptide.
  • the protein of interest may be an endogenous protein expressed from an endogenous gene in the host cell.
  • a "recombinant polypeptide” refers to a protein that is constructed or produced using recombinant DNA technology.
  • the protein of interest may be an exogenous sequence identical to the endogenous protein or a mutated version thereof, for example with attenuated biological activity, or fragment thereof, expressed from an exogenous vector.
  • the protein of interest may be a heterologous protein, not normally expressed by the host cell.
  • the protein of interest may be any suitable protein including a therapeutic, prophylactic or diagnostic protein.
  • the protein of interest is useful in the treatment of diseases or disorders including inflammatory diseases and disorders, immune disease and disorders, fibrotic disorders and cancers.
  • inflammatory disease or "disorder” and “immune disease or disorder” includes rheumatoid arthritis, psoriatic arthritis, still's disease, Muckle Wells disease, psoriasis, Crohn's disease, ulcerative colitis, SLE (Systemic Lupus Erythematosus), asthma, allergic rhinitis, atopic dermatitis, multiple sclerosis, vasculitis, Type I diabetes mellitus, transplantation and graft-versus-host disease.
  • fibrotic disorder includes idiopathic pulmonary fibrosis (IPF), systemic sclerosis (or scleroderma), kidney fibrosis, diabetic nephropathy, IgA nephropathy, hypertension, end-stage renal disease, peritoneal fibrosis (continuous ambulatory peritoneal dialysis), liver cirrhosis, age-related macular degeneration (ARMD), retinopathy, cardiac reactive fibrosis, scarring, keloids, burns, skin ulcers, angioplasty, coronary bypass surgery, arthroplasty and cataract surgery.
  • IPF idiopathic pulmonary fibrosis
  • systemic sclerosis or scleroderma
  • kidney fibrosis diabetic nephropathy
  • IgA nephropathy IgA nephropathy
  • hypertension end-stage renal disease
  • peritoneal fibrosis continuous ambulatory peritoneal dialysis
  • liver cirrhosis liver cirrhos
  • cancer includes a malignant new growth that arises from epithelium, found in skin or, more commonly, the lining of body organs, for example: breast, ovary, prostate, lung, kidney, pancreas, stomach, bladder or bowel. Cancers tend to infiltrate into adjacent tissue and spread (metastasise) to distant organs, for example: to bone, liver, lung or the brain.
  • the protein may be a proteolytically-sensitive polypeptide, i.e. proteins that are prone to be cleaved, susceptible to cleavage, or cleaved by one or more gram-negative bacterial, such as E. coli, proteases, either in the native state or during secretion.
  • the protein of interest is proteolytically-sensitive to the proteases DegP and Protease III. In one embodiment the protein of interest is proteolytically sensitive to the proteases DegP and Tsp. In one embodiment the protein of interest is proteolytically-sensitive to protease DegP.
  • the protein is a eukaryotic polypeptide.
  • the protein of interest expressed by the cells according to the invention may, for example be an immunogen, a fusion protein comprising two heterologous proteins or an antibody.
  • Antibodies for use as the protein of interest include monoclonal, multivalent, multi-specific, humanized, fully human or chimeric antibodies.
  • the antibody can be from any species but is preferably derived from a monoclonal antibody, a human antibody, or a humanized fragment.
  • the antibody can be derived from any class (e.g. IgG, IgE, IgM, IgD or IgA) or subclass of immunoglobulin molecule and may be obtained from any species including for example mouse, rat, shark, rabbit, pig, hamster, camel, llama, goat or human. Parts of the antibody fragment may be obtained from more than one species for example the antibody fragments may be chimeric. In one example the constant regions are from one species and the variable regions from another.
  • the antibody may be a complete antibody molecule having full length heavy and light chains or a fragment thereof, e.g. VH, VL, VHH, Fab, modified Fab, Fab', F(ab') 2 , Fv, scFv fragment, Fab-Fv, or a dual specificity antibody, such as a Fab-dAb, as described in PCT/GB2008/003331.
  • the antibody may be specific for any target antigen.
  • the antigen may be a cell- associated protein, for example a cell surface protein on cells such as bacterial cells, yeast cells, T-cells, endothelial cells or tumour cells, or it may be a soluble protein.
  • Antigens of interest may also be any medically relevant protein such as those proteins upregulated during disease or infection, for example receptors and/or their corresponding ligands.
  • cell surface proteins include adhesion molecules, for example integrins such as ⁇ ⁇ integrins e.g.
  • Soluble antigens include interleukins such as IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-12, IL-13, IL-14, IL- 16 or IL-17, such as IL17A and/or IL17F, viral antigens for example respiratory syncytial virus or cytomegalovirus antigens, immunoglobulins, such as IgE, interferons such as interferon a, interferon ⁇ or interferon ⁇ , tumour necrosis factor TNF (formerly known as tumour necrosis factor-a), tumor necrosis factor- ⁇ , colony stimulating factors such as G-CSF or G -CSF, and platelet derived growth factors such as PDGF-a, and PDGF- ⁇ and where appropriate receptors thereof.
  • Other antigens include bacterial cell surface antigens, bacterial toxins, viruses such as influenza, EBV, HepA, B and C, bioterrorism agents, radionuclides
  • the antibody may be used to functionally alter the activity of the antigen of interest.
  • the antibody may neutralize, antagonize or agonise the activity of said antigen, directly or indirectly.
  • the protein of interest expressed by the cells according to the present invention is an anti-TNF antibody, more preferably an anti-TNF Fab', as described in WOO 1/094585 (the contents of which are incorporated herein by reference).
  • the antibody molecule has specificity for human TNF (formerly known as TNFa), wherein the light chain comprises the light chain variable region of SEQ ID NO: 11 and the heavy chain comprises the heavy chain variable region of SEQ ID NO: 12.
  • the antibody molecule having specificity for human TNF is a Fab' and has a light chain sequence comprising or consisting of SEQ ID NO: 13 and a heavy chain sequence comprising or consisting of SEQ ID NO: 14.
  • Fab yield may be improved by expression in one or more cells according to the present invention.
  • the mutated DegP gene used in the strains of the present invention having chaperone activity and reduced protease activity improves Fab yield because the chaperone activity of DegP facilitates the correct folding of Fab.
  • antibody fragments may be further processed, for example by conjugation to another entity such as an effector molecule.
  • effector molecule includes, for example, antineoplastic agents, drugs, toxins (such as enzymatically active toxins of bacterial or plant origin and fragments thereof e.g. ricin and fragments thereof) biologically active proteins, for example enzymes, other antibody or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof e.g. DNA, RNA and fragments thereof, radionuclides, particularly radioiodide, radioisotopes, chelated metals, nanoparticles and reporter groups such as fluorescent compounds or compounds which may be detected by NMR or ESR spectroscopy.
  • toxins such as enzymatically active toxins of bacterial or plant origin and fragments thereof e.g. ricin and fragments thereof
  • biologically active proteins for example enzymes, other antibody or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof e.g. DNA, RNA and fragments thereof, radionuclides, particularly radioiodide, radioisotope
  • Effector molecular may be attached to the antibody or fragment thereof by any suitable method, for example an antibody fragment may be modified to attach at least one effector molecule as described in WO05/003171 or WO05/003170 (the contents of which are incorporated herein by reference).
  • WO05/003171 or WO05/003170 also describes suitable effector molecules.
  • the antibody or fragment thereof is PEGylated to generate a product with the required properties, for example similar to the whole antibodies, if required.
  • the antibody may be a PEGylated anti-TNF- a Fab', as described in WO01/094585, preferably having attached to one of the cysteine residues at the C-terminal end of the heavy chain a lysyl-maleimide-derived group wherein each of the two amino groups of the lysyl residue has covalently linked to it a methoxypoly(ethyleneglycol) residue having a molecular weight of about 20,000 Da, such that the total average molecular weight of the methoxypoly(ethyleneglycol) residues is about 40,O0ODa, more preferably the lysyl-maleimide-derived group is [1- [[[2-[[3-(2,5-dioxo-l-pyrrolidinyl)-l-oxoprop
  • the cell may also comprise further polynucleotide sequences encoding one or more further proteins of interest.
  • the polynucleotide encoding the protein of interest may be expressed as a fusion with another polypeptide, preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature polypeptide.
  • the heterologous signal sequence selected should be one that is recognized and processed by the host cell.
  • the signal sequence is substituted by a prokaryotic signal sequence. Suitable signal sequences include OmpA, PhoA, LamB, PelB, DsbA and DsbC.
  • an expression cassette is employed in the present invention to carry the polynucleotide encoding the protein of interest which typically comprises one or more protein coding sequences encoding one or more proteins of interest and one or more regulatory expression sequences.
  • the one or more regulatory expression sequences may include a promoter.
  • the one or more regulatory expression sequences may also include a 3 ' untranslated region such as a termination sequence. Suitable promoters are discussed in more detail below.
  • the cell according to the present invention comprises a vector, such as plasmid.
  • the vector preferably comprises one or more of the expression cassettes as defined above.
  • the vector for use in the present invention may be produced by inserting an expression cassette as defined above into a suitable vector.
  • the regulatory expression sequences for directing expression of the polynucleotide sequence encoding a protein of interest may be contained in the vector and thus only the encoding region of the polynucleotide may be required to complete the vector.
  • plasmid such as pBR322 or PACYC184, and/or
  • transposable genetic element such as a transposon
  • Such vectors usually comprise a plasmid origin of DNA replication, an antibiotic selectable marker, a promoter and transcriptional terminator separated by a multi-cloning site (expression cassette) and a DNA sequence encoding a ribosome binding site.
  • the promoters employed in the present invention can be linked to the relevant polynucleotide directly or alternatively be located in an appropriate position, for example in a vector such that when the relevant polypeptide is inserted the relevant promoter can act on the same.
  • the promoter is located before the encoding portion of the polynucleotide on which it acts, for example a relevant promoter before each encoding portion of polynucleotide. "Before" as used herein is intended to imply that the promoter is located at the 5 prime end in relation to the encoding polynucleotide portion.
  • the promoters may be endogenous or exogenous to the host cells. Suitable promoters include Lac, tac, tip, PhoA, Ipp, Arab, Tet and T7.
  • One or more promoters employed may be inducible promoters.
  • Expression units for use in bacterial systems also generally contain a Shine-Dalgarno (S. D.) ribosome binding sequence operably linked to the DNA encoding the polypeptide of interest.
  • a polynucleotide sequence comprises two or more encoding sequences for two or more proteins of interest, for example an antibody light chain and antibody heavy chain
  • the polynucleotide sequence may comprise one or more internal ribosome entry site (IRES) sequences which allows translation initiation in the middle of an mR A.
  • IRES sequence may be positioned between encoding polynucleotide sequences to enhance separate translation of the mRNA to produce the encoded polypeptide sequences.
  • the terminators may be endogenous or exogenous to the host cells.
  • a suitable terminator is rrnB.
  • transcriptional regulators including promoters and terminators and protein targeting methods may be found in "Strategies for Achieving High-Level Expression of Genes in Escherichia coi Savvas C. Makrides, Microbiological Reviews, Sept 1996, p 512-538.
  • Embodiments of the invention described herein with reference to the polynucleotide apply equally to alternative embodiments of the invention, for example vectors, expression cassettes and/or host cells comprising the components employed therein, as far as the relevant aspect can be applied to same.
  • the present invention also provides a method for producing a recombinant protein of interest comprising expressing the recombinant protein of interest in a recombinant gram-negative bacterial cell comprising a mutated DegP gene encoding a DegP protein having chaperone activity and reduced protease activity; and a mutated ptr gene encoding Protease III and/or a mutated Tsp gene encoding protease Tsp.
  • the gram negative bacterial cell and protein of interest preferably employed in the method of the present invention are described in detail above.
  • the present invention also provides a method for producing a recombinant heterologous protein of interest comprising expressing the recombinant protein of interest in a recombinant gram-negative bacterial cell comprising a mutated DegP gene encoding a DegP protein having chaperone activity and reduced protease activity.
  • the polynucleotide encoding the protein of interest may be incorporated into the host cell using any suitable means known in the art.
  • the polynucleotide is incorporated as part of an expression vector which is transformed into the cell.
  • the cell according to the present invention comprises an expression cassette comprising the polynucleotide encoding the protein of interest.
  • the polynucleotide sequence can be transformed into a cell using standard techniques, for example employing rubidium chloride, PEG or electroporation.
  • the method according to the present invention may also employ a selection system to facilitate selection of stable cells which have been successfully transformed with the polynucleotide encoding the protein of interest.
  • the selection system typically employs co-transformation of a polynucleotide sequence encoding a selection marker.
  • each polynucleotide transformed into the cell further comprises a polynucleotide sequence encoding one or more selection markers. Accordingly, the transformation of the polynucleotide encoding the protein of interest and the one or more polynucleotides encoding the marker occurs together and the selection system can be employed to select those cells which produce the desired proteins.
  • Cells able to express the one or more markers are able to survive/grow/multiply under certain artificially imposed conditions, for example the addition of a toxin or antibiotic, because of the properties endowed by the polypeptide/gene or polypeptide component of the selection system incorporated therein (e.g. antibiotic resistance). Those cells that cannot express the one or more markers are not able to survive/grow/multiply in the artificially imposed conditions.
  • the artificially imposed conditions can be chosen to be more or less vigorous, as required.
  • any suitable selection system may be employed in the present invention.
  • the selection system may be based on including in the vector one or more genes that provides resistance to a known antibiotic, for example a tetracycline, chloramphenicol, kanamycin or ampicillin resistance gene. Cells that grow in the presence of a relevant antibiotic can be selected as they express both the gene that gives resistance to the antibiotic and the desired protein.
  • the method according to the present invention further comprises the step of culturing the transformed cell in a medium to thereby express the protein of interest.
  • An inducible expression system or a constitutive promoter may be used in the present invention to express the protein of interest.
  • Suitable inducible expression systems and constitutive promoters are well known in the art.
  • the medium may be adapted for a specific selection system, for example the medium may comprise an antibiotic, to allow only those cells which have been successfully transformed to grow in the medium.
  • the cells obtained from the medium may be subjected to further screening and/or purification as required.
  • the method may further comprise one or more steps to extract and purify the protein of interest as required.
  • the polypeptide may be recovered from the strain, including from the cytoplasm, periplasm, or culture medium.
  • the specific method (s) used to purify a protein depends on the type of protein. Suitable methods include fractionation on immunoaffnity or ion-exchange columns; ethanol precipitation; reversed-phase HPLC; hydrophobic-interaction chromatography; chromatography on silica; chromatography on an ion-exchange resin such as S-SEPHAROSE and DEAE; chromatofocusing; ammonium-sulfate precipitation; and gel filtration.
  • Antibodies may be suitably separated from the culture medium and/or cytoplasm extract and/or periplasm extract by conventional antibody purification procedures such as, for example, protein A-Sepharose, protein G chromatography, protein L chromatograpy, thiophilic, mixed mode resins, Histag, FLAGTag, hydroxy lapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, Ammonium Sulphate, ethanol or PEG fractionation/precipitation, ion exchange membranes, expanded bed adsorption chromatography (EBA) or simulated moving bed chromatography.
  • EBA expanded bed adsorption chromatography
  • the method may also include a further step of measuring the quantity of expression of the protein of interest and selecting cells having high expression levels of the protein of interest.
  • One or more method steps described herein may be performed in combination in a suitable container such as a bioreactor.
  • the invention also extends to use of a recombinant polynucleotide sequence encoding a DegP protein having chaperone activity and reduced protease activity for expressing a heterologous protein of interest.
  • the invention provides use of a recombinant polynucleotide sequence encoding a DegP protein having chaperone activity and reduced protease activity in one or more expression cassettes, one or more vectors or one or more cells as described above for expressing a heterologous protein of interest.
  • W31 10 genotype F- LAM- IN (rrnD-rrnE)l rphl (ATCC no. 27325).
  • W31 10A as shown in the figures, is a different batch of W31 10.
  • the mutant E. coli cell strain MXE005 having genotype ATsp, DegP S210A was generated using a gene replacement vector system using the pK03 homologous recombination/replacement plasmid (Link et al., 1997, Journal of Bacteriology, 179,
  • Tsp and DegP integration cassettes were moved as Sal I, Not I restriction fragments into similarly restricted pK03 plasmids.
  • the plasmid uses the temperature sensitive mutant of the pSClOl origin of replication (RepA) along with a chloramphenicol marker to force and select for chromosomal integration events.
  • the sacB gene which encodes for levansucrase is lethal to E . coli grown on sucrose and hence (along w r ith the chloramphenicol marker and pSClOl origin) is used to force and select for de-integration and plasmid curing events.
  • This methodology had been described previously (Hamilton et al., 1989, Journal of Bacteriology, 171 , 4617-4622 and Blomfield et al., 1991, Molecular Microbiology, 5, 1447-1457).
  • the pK03 system removes all selective markers from the host genome except for the inserted gene.
  • pMXE191 comprising the knockout mutated Tsp gene as shown in the SEQ ID NO: 3 comprising EcoR I and Ase I restriction markers.
  • pMXE192 comprising the mutated DegP gene as shown in the SEQ ID NO: 9 comprising an Ase I.
  • E. coli W3110 cells prepared using the method found in Chung CT et al Transformation and storage of bacterial cells in the same solution.
  • Day 1 40 ⁇ 1 of E.coli cells were mixed with (lOpg) ⁇ of p 03 DNA in a chilled BioRad 0.2cm electroporation cuvette before electroporation at 2500V, 25 ⁇ and 200 ⁇ .
  • ⁇ of 2xPY was added immediately, the cells recovered by shaking at 250rpm in an incubator at 30 U C for 1 hour.
  • Cell strain MXE005 was tested to confirm successful modification of genomic DNA carrying the mutated protease genes by PCR amplification of the region of each mutated protease gene comprising a non-naturally occurring Ase I restriction site, as shown in Figures 10b and 10c, using oligonucleotides primers. The amplified regions of the DNA were then analyzed by gel electrophoresis before and after incubation with Ase I restriction enzyme to confirm the presence of the non-naturally occurring Ase I restriction site in the mutated genes. This method was carried out as follows:
  • oligos were used to amplify, using PCR, genomic DNA from prepared E. coli cell lysate from MXE005 and W31 10:
  • the lysates were prepared by heating a single colony of cells for 10 minutes at 95 °C in 20ul of lx PCR buffer. The mixture was allowed to cool to room temperature then centrifugation at 13,200rpm for 10 minutes. The supernatant was removed and labeled as 'cell lysate'.
  • the MXE005 strain was amplified using the Tsp pair and DegP pair of oligonucleotides primers.
  • the DNA was amplified using a standard PCR procedure.
  • the genomic fragments amplified showed the correct sized band of 2.8Kb for Tsp and 2.2K.b for DegP.
  • Plasmid pMXE1 17 (pTTO CDP870 or 40.4), an expression vector for the CDP870 Fab' (an anti-TNF Fab' having SEQ ID NO: 13 and 14), was constructed using conventional restriction cloning methodologies which can be found in Sambrook et al 1989, Molecular cloning: a laboratory manual. CSHL press, N.Y.
  • the plasmid pMXE1 17 contained the following features; a strong tac promoter and lac operator sequence.
  • the Fab light and heavy chain genes were transcribed as a single dicistronic message. DNA encoding the signal peptide from the E.
  • coli OmpA protein was fused to the 5' end of both light and heavy chain gene sequences, which directed the translocation of the polypeptides to the E. coli periplasm. Transcription was terminated using a dual transcription terminator rmB tlt2.
  • the laclq gene encoded the constitutively expressed Lac I repressor protein. This repressed transcription from the tac promoter until de-repression was induced by the presence of allolactose or IPTG.
  • the origin of replication used was pl5A, which maintained a low copy number.
  • the plasmid contained a tetracycline resistance gene for antibiotic selection.
  • pMXE1 17 was then transformed into chemically competent proteases deficient cells (strain MXE005) and W3110 cells prepared using the method found in Chung C.T et al Transformation and storage of bacterial cells in the same solution. PNAS 86:2172- 2175 (1989).
  • Example 2 Expression of an anti-TNFa Fab' in mutated E. coli strain using shake flask cultures
  • Shake flask cultures were initiated by addition of a 2ml aliquot of thawed defined medium 'adapted cells' to 200ml of SM6E media plus tetracycline l Oug/ml. These where grown overnight at 30°C with agitation at 250rpm. Each strain being tested was grown in triplicate.
  • Figure 1 shows growth of MXE005 compared to the wild type W3110.
  • Figure 2 shows improved expression of the Fab' from MXE005 compared to the wild type W3110.
  • Example 3 Expression of an anti-mIL13 mouse Fab in mutated E. coli strain using shake flask cultures
  • Strain MXE005 and wild type W3110 cells were transformed with plasmid pMKC006 expressing a murinised anti-mIL13 Fab' and tested using the same shake flask method described in Example 2 except the experiment was stopped after 6 hours instead of 24 hours.
  • FIG. 3 shows the expression of an anti-mIL-13 mouse Fab in MXE005 and W31 10
  • MXE005 shows higher Fab expression compared to W31 10.
  • the anti-TNFa Fab' was immobilised onto CM5 sensor chips using standard NHS EDC chemistry. Residual NHS esters were inactivated with ethanolamine hydrochloride (1 M).
  • Fab' fragments were captured by either an immobilised monoclonal antiheavy chain or by an immobilised monoclonal anti-light chain antibody in separate flow cells.
  • the presence of bound Fab' was revealed by binding of the complementary monoclonal antibody (anti-light chain or anti-heavy chain) in a second step.
  • High levels of immobilised antibody ensure that measurements are performed under mass transport- limited conditions, where the contribution of the association rate constant to binding is low in comparison to the contribution made by the concentration of the Fab' in the sample.
  • the solution phase monoclonal antibody used in the second step is passed over the surface at a high concentration so that binding is not limited by the association rate constant of this interaction.
  • Figure 4 shows the light chain (L chain), heavy chain (H chain) and Fab' expression during the course of a fermentation run where a higher light chain after 6 hours from MXE005 compared to W31 10 is shown.
  • Figure 4 also shows higher Fab' expression from MXE005 after 2 hours, 4 hours and 6 hours compared to W3100.
  • a polyclonal rabbit anti-human Fab' sera (UCB) was applied at a dilution of 1 in 1000 in 5mls of blocking buffer and incubated at room temperature for 1 hour with gentle agitation. The membrane was washed three times for 5mins each with gentle agitation with blocking buffer. A secondary antibody (donkey anti-rabbit IgG HRP conjugated antibody (Jackson)) applied at a dilution of 1 in 5000 in blocking buffer and incubation at room temperature for 1 hour with gentle agitation. The membrane was washed four times for 5 minutes each with agitation firstly with blocking buffer followed by PBS, 0.1% Tween for two washes then PBS for the final wash. The blot was visualized using Metal Enhanced Dab substrate (Thermo Scientific).
  • Example 6 Growth of mutated E. coli strain and expression of Fab' in mutated E. coli strain using high density fermentations
  • Strain MXE005 and wild type W31 10 cells were transformed with plasmid pMXEl 17 tested in fermentation experiments comparing growth and expression of an anti-TNFa Fab'.
  • the fermentation growth medium was based on SM6E medium (described in Humphreys et al., 2002, Protein Expression and Purification, 26, 309-320) with 3.86 g/1 NaH 2 P0 4 .H 2 0 and 1 12 g/1 glycerol.
  • Inoculum Inoculum cultures were grown in the same medium supplemented with 10 ⁇ g/ml tetracycline. Cultures were incubated at 30°C with agitation for approximately 22 hours.
  • Fermentation Fermentation. Fermenters (2.5 litres total volume) were seeded with inoculum culture to 0.3-0.5 OD 6 o 0 . Temperature was maintained at 30°C during the growth phase and was reduced to 25°C prior to induction. The dissolved oxygen concentration was maintained above 30% air saturation by variable agitation and airflow. Culture pH was controlled at 7.0 by automatic titration with 15% (v/v) NH 4 OH and 10% (v/v) cone. H 2 S0 4 . Foaming was controlled by the addition of 10% (v/v) Struktol J673 solution (Schill and Seilacher).
  • Biomass concentration was determined by measuring the optical density of cultures at 600 nm.
  • Fab' quantification Fab' concentrations in periplasmic extracts and culture supernatants were determined by Fab' assembly ELISA as described in Humphreys et al., 2002, Protein Expression and Purification, 26, 309-320.
  • Figure 6 shows the growth profile of MXE005 compared to control W31 10.
  • the growth profile of MXE005 is faster over the initial period of approximately 35 hours compared to the control W3110.
  • Figure 7 shows Fab' yields from the supernatant (dotted lines) and periplasm (solid lines) of E. coli strain MXE005 and the W31 10 control.
  • the MXE005 strain shows higher Fab' yield from the periplasm for approximately 28 hours compared to the control and significantly higher supernatant Fab' yield compared to the control over the whole fermentation period.
  • Figure 8 shows the total Fab' yield from the supernatant and periplasm of the E. coli strain MXE005 where it can be clearly seen that the MXE005 strain produced significantly higher yield compared to the control.
  • Figure 9 shows the Fab' specific production rate of E. coli strain MXE005 and the W31 10 control where it can be seen that MXE005 has a significantly higher specific production rate compared to W31 10.

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Abstract

La présente invention concerne une cellule bactérienne gram-négative comprenant : a) un gène DegP muté codant pour une protéine DegP qui a une activité chaperonne et a une activité protéase réduite ; et b) un gène Tsp muté, le gène Tsp muté codant pour une protéine Tsp ayant une activité protéase réduite ou étant un gène Tsp muté inactivé ; et/ou c) un gène ptr muté, le gène ptr muté codant pour une protéine protéase III ayant une activité protéase réduite ou étant un gène ptr muté inactivé.
PCT/GB2010/001792 2009-09-24 2010-09-23 Souche bactérienne pour l'expression de protéines recombinantes, ayant une degp à activité protéase déficiente et conservant une activité chaperonne et des gènes tsp et ptr inactivés WO2011036455A1 (fr)

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WO2017132298A1 (fr) 2016-01-27 2017-08-03 Medimmune, Llc Procédé de préparation d'anticorps ayant un profil de glycosylation défini
CN113874518A (zh) * 2019-03-18 2021-12-31 生物辐射Abd瑟罗泰克有限公司 保护含SpyTag的周质融合蛋白免于蛋白酶Tsp和OmpT降解
EP3988107A1 (fr) * 2015-10-30 2022-04-27 Synlogic Operating Company, Inc. Bactéries modifiées pour traiter des maladies pour lesquelles une diminution de l'inflammation intestinale et/ou une plus grande imperméabilité de la muqueuse intestinale s'avèrent bénéfiques

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Cited By (3)

* Cited by examiner, † Cited by third party
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EP3988107A1 (fr) * 2015-10-30 2022-04-27 Synlogic Operating Company, Inc. Bactéries modifiées pour traiter des maladies pour lesquelles une diminution de l'inflammation intestinale et/ou une plus grande imperméabilité de la muqueuse intestinale s'avèrent bénéfiques
WO2017132298A1 (fr) 2016-01-27 2017-08-03 Medimmune, Llc Procédé de préparation d'anticorps ayant un profil de glycosylation défini
CN113874518A (zh) * 2019-03-18 2021-12-31 生物辐射Abd瑟罗泰克有限公司 保护含SpyTag的周质融合蛋白免于蛋白酶Tsp和OmpT降解

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