Bacterial host strain

Patent metadata

Field	Value
Publication number	US-9315770-B2
Application number	US-201514827408-A
Country	US
Kind code	B2
Filing date	Aug 17, 2015
Priority date	Sep 24, 2009
Publication date	Apr 19, 2016
Grant date	Apr 19, 2016

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

The invention claimed is:

1. A mutant E. coli cell strain selected from:
MXE002 having genotype Δptr and deposited with the National Collection of Type Cultures, MPA, United Kingdom, under Accession number NCTC13445;
MXE003 having genotype DegP S210A and deposited with the National Collection of Type Cultures, HPA, United Kingdom, under Accession number NCTC13446; or
MXE006 having genotype Δptr, DegP S210A and deposited with the National Collection of Type Cultures, HPA, United Kingdom, under Accession number NCTC13449.

2. An isolated gram-negative bacterial cell comprising 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:
a) 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;
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
c) a polynucleotide encoding a protein of interest.

3. The isolated gram-negative bacterial cell according to claim 2 , wherein said 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.

4. The isolated gram-negative bacterial cell according to claim 2 , wherein one or more of His105, Asp135, and Ser210 are substituted with a glycine or alanine.

5. The isolated gram-negative bacterial cell according to claim 3 , wherein His105, Asp135, and Ser210 are substituted with a glycine or alanine.

6. The isolated gram-negative bacterial cell according to claim 2 , wherein His105 is substituted with a glycine, alanine, valine or leucine.

7. The isolated gram-negative bacterial cell according to claim 2 , wherein Asp135 is substituted with a glycine, alanine, lysine or arginine.

8. The isolated gram-negative bacterial cell according to claim 2 , wherein Ser210 is substituted with a glycine, alanine, valine, leucine, phenylalanine or tyrosine.

9. The isolated gram-negative bacterial cell according to claim 3 , 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.

10. The isolated gram-negative bacterial cell according to claim 2 , wherein said bacterial cell comprises 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.

11. The isolated gram-negative bacterial cell according to claim 2 , 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.

12. The isolated gram-negative bacterial cell according to claim 2 , wherein said bacterial cell comprises a mutated DegP gene and a polynucleotide encoding a protein of interest.

13. The isolated gram-negative bacterial cell according to claim 2 , wherein said bacterial cell comprises 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.

14. The isolated gram-negative bacterial cell according to claim 10 , wherein said bacterial cell further comprises a polynucleotide encoding a protein of interest.

15. The isolated gram-negative bacterial cell according to claim 11 , wherein said bacterial cell further comprises a polynucleotide encoding a protein of interest.

16. The isolated gram-negative bacterial cell according to claim 13 , wherein said bacterial cell further comprises a polynucleotide encoding a protein of interest.

17. The isolated gram-negative bacterial cell according to claim 12 , wherein said protein of interest is an antibody or antigen-binding fragment thereof.

18. The isolated gram-negative bacterial cell according to claim 14 , wherein said protein of interest is an antibody or antigen-binding fragment thereof.

19. The isolated gram-negative bacterial cell according to claim 15 , wherein said protein of interest is an antibody or antigen-binding fragment thereof.

20. The isolated gram-negative bacterial cell according to claim 16 , wherein said protein of interest is an antibody or antigen-binding fragment thereof.

Assignees

• Ucb Pharma Sa

Inventors

• Ellis Mark
• Humphreys David Paul

Classifications

• C12N9/52

  derived from bacteria {or Archaea} · CPC title

• C12N15/70

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

• C12N1/20 Primary

  Bacteria; Culture media therefor · CPC title

• C07K16/241 Primary

  Tumor Necrosis Factors · CPC title

• C12P21/02

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