Bacterial host strain comprising a mutant SPR gene and a wild-type TSP gene

Patent metadata

Field	Value
Publication number	US-9493558-B2
Application number	US-201514633257-A
Country	US
Kind code	B2
Filing date	Feb 27, 2015
Priority date	Jan 14, 2010
Publication date	Nov 15, 2016
Grant date	Nov 15, 2016

Abstract

The present invention provides a recombinant gram-negative bacterial cell comprising a mutant spr gene encoding a mutant spr protein and wherein the cell comprises a non-recombinant wild-type chromosomal Tsp gene.

First claim

The invention claimed is:

1. A recombinant gram-negative bacterial cell comprising a mutant spr gene encoding a mutant spr protein containing a mutation that is an amino acid substitution at one or more amino acids within the spr protein, wherein the cell comprises a non-recombinant wild-type chromosomal Tsp gene and expression of said mutant spr gene causes the cell to exhibit increased yield of a recombinant protein of interest or reduced lysis as compared to a wild-type bacterial cell containing a wild-type spr gene and expressing a protein of interest and, optionally, one or more of:
a) a mutated DegP gene encoding a DegP protein having chaperone activity and reduced protease activity;
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;
c) a mutated OmpT gene, wherein the mutated OmpT gene encodes an OmpT protein having reduced protease activity or is a knockout mutated OmpT gene;
d) a recombinant polynucleotide encoding DsbC; and
e) a polynucleotide encoding a protein of interest.

2. The cell according to claim 1 , wherein the mutant spr gene encodes a mutant spr protein having a mutation at one or more amino acids selected from the group consisting of H145, C94, and H157A, wherein the wild-type spr protein has the amino acid sequence of SEQ ID NO: 21.

3. The cell according to claim 2 , wherein the mutant spr gene encodes a mutant spr protein having one or more mutations selected from the group consisting of H145A, C94A, and H157A, wherein the wild-type spr protein has the amino acid sequence of SEQ ID NO: 21.

4. The cell according to claim 3 , wherein the one or more spr mutations is C94A.

5. The cell according to claim 3 , wherein the one or more spr mutations is H145A.

6. The cell according to claim 3 , wherein the one or more spr mutations is H157A.

7. The cell according to claim 3 , wherein the one or more spr mutations is C94A and H145A.

8. The cell according to claim 3 , wherein the one or more spr mutations is C94A and H157A.

9. The cell according to claim 3 , wherein the one or more spr mutations is C94A H145A, and H157A.

10. The cell according to claim 2 , wherein the cell comprises a vector comprising a polynucleotide encoding DsbC and the polynucleotide encoding the protein of interest.

11. The cell according to claim 10 , wherein the vector comprises a promoter which controls the expression of both the recombinant polynucleotide encoding DsbC and the polynucleotide sequence encoding a protein of interest.

12. The cell according to claim 1 , wherein the cell comprises a recombinant polynucleotide encoding DsbC.

13. The cell according to claim 1 , wherein the cell further comprises one or more of the following mutated genes:
a) a mutated DegP gene encoding a DegP protein having chaperone activity and reduced protease activity;
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 OmpT gene, wherein the mutated OmpT gene encodes an OmpT protein having reduced protease activity or is a knockout mutated OmpT gene.

14. The cell according to claim 1 , wherein the cell is E. coli.

15. The cell according to claim 1 , wherein the cell further comprises a polynucleotide encoding a protein of interest.

16. The cell according to claim 15 , wherein the protein of interest is an antibody or an antigen binding fragment thereof.

17. The cell according to claim 16 , wherein the antibody or antigen binding fragment thereof is specific for TNF.

18. The cell according to claim 17 , wherein the cell comprises a recombinant polynucleotide encoding DsbC.

19. A method for producing a recombinant protein of interest comprising culturing the recombinant gram-negative bacterial cell as defined in claim 1 that comprises a polynucleotide encoding a recombinant protein of interest in a culture medium under conditions effective to express the recombinant protein of interest and recovering the recombinant protein of interest from the periplasm of the recombinant gram-negative bacterial cell and/or the culture medium.

20. The method according to claim 19 , wherein the recombinant protein of interest is recovered from the periplasm and/or the supernatant obtained by separating the cultured recombinant bacterial cell from the culture medium.

21. The method according to claim 19 , wherein the cell comprises a recombinant polynucleotide encoding DsbC and the cell is cultured under conditions effective to express the recombinant polynucleotide encoding DsbC.

22. The method according to claim 21 , wherein the expression of the polynucleotide encoding the protein of interest and the recombinant polynucleotide encoding DsbC is induced by adding an inducer to the culture medium, wherein the polynucleotide encoding the protein of interest and the polynucleotide encoding DsbC are under the control of a promoter inducible by the inducer.

23. The method according to claim 21 , wherein the method further comprises separating the recombinant protein of interest from DsbC.

24. The method according to claim 19 , wherein the protein of interest is an antibody or antigen binding fragment of an antibody specific for TNF.

Assignees

• Ucb Pharma Sa

Inventors

• Ellis Mark
• Humphreys David Paul

Classifications

• C12P21/02

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

• C07K2317/14

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

• C07K2317/55

  Fab or Fab' · CPC title

• C12N15/70 Primary

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

• C12N1/00

  Microorganisms; Compositions thereof (medicinal preparations containing material from protozoa, bacteria or viruses A61K35/66, from algae A61K36/02, from fungi A61K36/06; preparing medicinal bacterial antigen or antibody compositions, e.g. bacterial vaccines, A61K39/00); Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor · CPC title