Bacterial host strain expressing recombinant DsbC and having reduced Tsp activity

Patent metadata

Field	Value
Publication number	US-9493559-B2
Application number	US-201514633294-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, characterized in that the cell comprises a recombinant polynucleotide encoding DsbC and has reduced Tsp protein activity compared to a wild-type cell.

First claim

The invention claimed is:

1. A recombinant gram-negative bacterial cell, wherein the cell:
a) comprises an exogenous recombinant polynucleotide encoding DsbC; and
b) has reduced Tsp protein activity as compared to a wild-type cell resulting from a mutation in the Tsp gene that encodes a mutated Tsp protein having reduced protease activity or a mutation in the Tsp gene or regulatory sequence of the Tsp gene that reduces or eliminates expression of the Tsp protein, and optionally, one or more of:
a) 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 mutant spr protein reduces lysis of cells containing a mutated Tsp gene;
b) a mutated DegP gene encoding a DegP protein having chaperone activity and reduced protease activity;
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;
d) a mutated OmpT gene, wherein the mutated OmpT gene encodes a OmpT protein having reduced protease activity or is a knockout mutated OmpT gene; and
e) a polynucleotide sequence encoding a protein of interest.

2. The cell according to claim 1 , wherein the cell comprises the mutant spr gene encoding a mutant spr protein that reduces lysis of cells containing a mutated Tsp gene.

3. The cell according to claim 2 , 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 H157, H145, and C94, wherein the wild-type spr protein has the sequence of SEQ ID NO: 21.

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

5. The cell according to claim 4 , wherein the mutant spr gene encodes a mutant spr protein and said one or more mutations is H157A.

6. The cell according to claim 4 , wherein the mutant spr gene encodes a mutant spr protein and said one or more mutations is H145A.

7. The cell according to claim 4 , wherein the mutant spr gene encodes a mutant spr protein and said one or more mutations is C94A.

8. The cell according to claim 4 , wherein the mutant spr gene encodes a mutant spr protein and said one or more mutations are H157A and H145A.

9. The cell according to claim 4 , wherein the mutant spr gene encodes a mutant spr protein and said one or more mutations are H157A and C94A.

10. The cell according to claim 4 , wherein the mutant spr gene encodes a mutant spr protein and said one or more mutations are H145A and C94A.

11. The cell according to claim 4 , wherein the mutant spr gene encodes a mutant spr protein and said one or more mutations are H157A, H145A, and C94A.

12. The cell according to claim 1 , wherein the cell comprises one or more of the following:
a) the mutated DegP gene encoding the DegP protein having chaperone activity and reduced protease activity;
b) the mutated ptr gene, wherein the mutated ptr gene encodes the Protease III protein having reduced protease activity or is the knockout mutated ptr gene; and
c) the mutated OmpT gene, wherein the mutated OmpT gene encodes the OmpT protein having reduced protease activity or is a knockout mutated OmpT gene.

13. The cell according to claim 1 , wherein the mutation in the Tsp gene that eliminates expression of the Tsp protein comprises a knockout mutation of the Tsp gene.

14. The cell according to claim 13 , wherein the cell's genome is isogenic to a wild-type bacterial cell from which the cell is derived except for the mutated Tsp gene, the recombinant polynucleotide encoding DsbC and optionally, the mutated spr gene.

15. The cell according to claim 13 , wherein the knockout mutation of the Tsp gene comprises a mutation to the Tsp gene start codon and/or one or more stop codons positioned downstream of the gene start codon and upstream of the Tsp gene stop codon.

16. The cell according to claim 15 , wherein the knockout mutation of the Tsp gene comprises a restriction marker site created by a missense mutation to the Tsp gene start codon and optionally, one or more additional point mutations.

17. The cell according to claim 16 , wherein the knockout mutated Tsp gene comprises SEQ ID NO:3.

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

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

20. The cell according to claim 19 , wherein the cell comprises a vector, said vector comprising both the recombinant polynucleotide encoding DsbC and the polynucleotide encoding the protein of interest.

21. The cell according to claim 20 , wherein the vector comprises a promoter which controls the expression of the recombinant polynucleotide encoding DsbC and the polynucleotide sequence encoding a protein of interest.

22. The cell according to claim 19 , wherein the protein of interest is an antibody or an antigen binding fragment thereof.

23. The cell according to claim 22 , wherein the antibody or antigen binding fragment thereof is specific for TNF.

24. A method for producing a protein of interest comprising:
a) culturing a recombinant gram-negative bacterial cell as defined in claim 2 that comprises a polynucleotide encoding a protein of interest in a culture medium under conditions effective to express the recombinant protein of interest and the recombinant polynucleotide encoding DsbC; and
b) recovering the protein of interest from the periplasm of the recombinant gram-negative bacterial cell and/or the culture medium.

25. The method according to claim 24 , wherein the expression of the polynucleotide sequence encoding a 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.

26. The method according to claim 24 , wherein the method further comprises separating the protein of interest from DsbC.

Assignees

• Ucb Pharma Sa

Inventors

• Ellis Mark
• Humphreys David Paul

Classifications

• C07K14/245 Primary

  Escherichia (G) · CPC title

• C12N1/20

  Bacteria; Culture media therefor · CPC title

• C07K16/241 Primary

  Tumor Necrosis Factors · CPC title

• C07K2317/14

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

• C12Y503/04001

  Protein disulfide-isomerase (5.3.4.1), i.e. disufide bond-forming enzyme · CPC title