Bacterial host strain

Patent metadata

Field	Value
Publication number	US-9550973-B2
Application number	US-201615083446-A
Country	US
Kind code	B2
Filing date	Mar 29, 2016
Priority date	Sep 24, 2009
Publication date	Jan 24, 2017
Grant date	Jan 24, 2017

Abstract

A recombinant gram-negative bacterial cell comprising one or more of the following mutated protease genes: a) a mutated Tsp gene, wherein the mutated Tsp gene encodes a Tsp protein having reduced protease activity or is a knockout mutated Tsp gene; b) 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 c) a mutated DegP gene encoding a DegP protein having chaperone activity and reduced protease activity; wherein the cell is isogenic to a wild-type bacterial cell except for the mutated Tsp gene and/or mutated ptr gene and/or mutated DegP gene and optionally a polynucleotide sequence encoding a protein of interest.

First claim

We claim:

1. A method for producing a recombinant protein of interest comprising expressing a polynucleotide encoding the recombinant protein of interest in a recombinant gram-negative bacterial cell selected from the group consisting of:
a) a mutant E. coli cell strain identified as MXE002 having genotype Δptr and deposited with the National Collection of Type Cultures, HPA, United Kingdom, under Accession Number NCTC13445;
b) a mutant E. coli cell strain identified as MXE003 having genotype DegP S210A and deposited with the National Collection of Type Cultures, HPA, United Kingdom, under Accession Number NCTC13446; and
c) a mutant E. coli cell strain identified as MXE006 having genotype Δptr, DegP S210A and deposited with the National Collection of Type Cultures, HPA, United Kingdom, under Accession Number NCTC13449.

2. The method according to claim 1 , wherein said recombinant protein of interest is an antibody or antigen-binding fragment thereof.

3. The method according to claim 2 , wherein said antibody or antigen-binding fragment thereof specifically binds tumour necrosis factor (TNF).

4. A method for producing a recombinant protein of interest comprising expressing a polynucleotide encoding the recombinant protein of interest in a recombinant gram-negative bacterial cell comprising said polynucleotide encoding a protein of interest, a mutated DegP gene, wherein the mutated DegP gene encodes a DegP protease that comprises a mutation at an amino acid selected from the group consisting of His105, Asp135, Ser210 and combinations thereof and said bacterial cell is isogenic to a wild-type bacterial cell except for the mutated DegP gene and, optionally, one or more of the following genetic alterations:
i) 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/or
ii) a mutated Tsp gene, wherein the mutated Tsp gene encodes a Tsp protein having reduced protease activity or is a knockout mutated Tsp gene.

5. The method according to claim 4 , wherein the DegP protease comprises: a mutation to His105; a mutation to Asp135; a mutation to Ser210; a mutation to His105 and Asp135; a mutation to His105 and Ser210; a mutation to Asp135 and Ser210; or a mutation to His105, Asp135 and Ser210.

6. The method according to claim 5 , wherein one or more of His105, Asp135, and Ser210 are substituted with a glycine or alanine.

7. The method according to claim 5 , wherein His105, Asp135, and Ser210 are substituted with a glycine or alanine.

8. The method according to claim 5 , wherein His105 is substituted with a glycine, alanine, valine or leucine.

9. The method according to claim 5 , wherein Asp135 is substituted with a glycine, alanine, lysine or arginine.

10. The method according to claim 5 , wherein Ser210 is substituted with a glycine, alanine, valine, leucine, phenylalanine or tyrosine.

11. The method according to claim 5 , wherein His105 is substituted with a glycine, alanine, valine or leucine; Asp135 is substituted with a glycine, alanine, lysine or arginine; and Ser210 is substituted with a glycine, alanine, valine, leucine, phenylalanine or tyrosine.

12. The method according to claim 4 , wherein said bacterial cell comprises said polynucleotide encoding said protein of interest, a mutated DegP gene 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.

13. The method according to claim 4 , wherein said bacterial cell comprises a mutated DegP 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.

14. The method according to claim 4 , wherein said bacterial cell comprises said polynucleotide encoding said protein of interest and a mutated DegP gene.

15. The method according to claim 4 , wherein said bacterial cell comprises said polynucleotide encoding said protein of interest, a mutated DegP gene, 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.

16. The method according to claim 4 , wherein said protein of interest is an antibody or antigen-binding fragment thereof.

17. The method according to claim 12 , wherein said protein of interest is an antibody or antigen-binding fragment thereof.

18. The method according to claim 13 , wherein said protein of interest is an antibody or antigen-binding fragment thereof.

19. The method according to claim 14 , wherein said protein of interest is an antibody or antigen-binding fragment thereof.

20. The method according to claim 15 , wherein said recombinant protein of interest is an antibody or antigen-binding fragment thereof.

21. The method according to claim 16 , wherein said antibody or antigen-binding fragment thereof specifically binds tumour necrosis factor (TNF).

22. The method according to claim 17 , wherein said antibody or antigen-binding fragment thereof specifically binds tumour necrosis factor (TNF).

23. The method according to claim 18 , wherein said antibody or antigen-binding fragment thereof specifically binds tumour necrosis factor (TNF).

24. The method according to claim 19 , wherein said antibody or antigen-binding fragment thereof specifically binds tumour necrosis factor (TNF).

25. The method according to claim 20 , wherein said antibody or antigen-binding fragment thereof specifically binds tumour necrosis factor (TNF).

Assignees

• Ucb Pharma Sa

Inventors

• Ellis Mark
• Humphreys David Paul

Classifications

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C07K16/244

  Interleukins [IL] · CPC title

• C07K2317/14

  Specific host cells or culture conditions, e.g. components, pH or temperature · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C07K2317/55

  Fab or Fab' · CPC title