Expression of mammalian proteins in Pseudomonas fluorescens

• US 10,041,102 B2
• Filed: 08/05/2016
• Issued: 08/07/2018
• Est. Priority Date: 10/08/2002
• Status: Expired due to Term

• Current Assignee: Pelican Technology Holdings, Inc (Primordial Genetics Inc.)
• Original Assignee: Pfenex Inc.
• Inventors: Retallack, Diane M., Squires, Charles H., Watkins, David C., Lee, Stacey L., Gaertner, Frank H., Shutter, Robert
• Primary Examiner(s): Hibbert, Catherine S
• Application Number:
  US15/230,192
• Publication Number:
  US 20170051327A1
• Time in Patent Office: 732 Days
• Field of Search: None
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• CPC Class Codes: C07K 14/57 IFN-gamma
• CPC Class Codes: C07K 14/61 Growth hormone [GH], i.e. somatotropin
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 2317/14 Specific host cells or culture conditions, e.g. components, pH or temperature
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C12N 15/78 for Pseudomonas
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: C12Y 302/01023 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase

Abstract

The invention is a process for improved production of a recombinant mammalian protein by expression in a Pseudomonad, particularly in a Pseudomonas fluorescens organism. The process improves production of mammalian proteins, particularly human or human-derived proteins, over known expression systems such as E. coli in comparable circumstances. Processes for improved production of isolated mammalian, particularly human, proteins are provided.

Claims

1. A process for increasing expression of a recombinant cytokine or chemokine comprising:

(a) growing a Pseudomonas fluorescens (P. fluorescens) host cell transformed with a nucleic acid comprising a sequence encoding the recombinant cytokine or chemokine under conditions that allow expression of the recombinant cytokine or chemokine, wherein the nucleic acid does not comprise a secretion signal coding sequence; and

(b) isolating the recombinant cytokine or chemokine expressed;

wherein the recombinant cytokine or chemokine is produced in the cytoplasm of the P. fluorescenshost cell as 5-40% total cell protein, wherein the recombinant cytokine or chemokine produced is present in the host cell in a soluble form, and wherein the soluble recombinant cytokine or chemokine is produced at a titre in the P. fluorescens host cell that is about 1.5-fold to about 8-fold higher than the titre of the soluble recombinant cytokine or chemokine produced in an E. coli host cell.

2. The process of claim 1, further comprising substantially purifying the recombinant cytokine or chemokine isolated in step (b).

3. The process of claim 1, wherein the recombinant cytokine or chemokine produced is present in the host cell in an active form.

4. The process of claim 1, wherein the recombinant cytokine or chemokine is a human peptide.

5. The process of claim 1 wherein the recombinant cytokine or chemokine is produced as about 10% to 40% total cell protein.

6. The process of claim 1 wherein the recombinant cytokine or chemokine is produced as about 15% to 40% total cell protein.

7. The process of claim 1 wherein the recombinant cytokine or chemokine is produced as about 20% to 40% total cell protein.

8. The process of claim 1 wherein the recombinant cytokine or chemokine is produced as about 30% to 40% total cell protein.

9. The process of claim 1, wherein the soluble recombinant cytokine or chemokine is produced at a titre in the P. fluorescens host cell that is about 2-fold to about 8-fold higher than the titre of the soluble recombinant cytokine or chemokine produced in an E. coli host cell.

10. The process of claim 1, wherein the soluble recombinant cytokine or chemokine is produced at a titre in the P. fluorescens host cell that is about 3-fold to about 8-fold higher than the titre of the soluble recombinant cytokine or chemokine produced in an E. coli host cell.

11. A process for producing a recombinant cytokine or chemokine in a P. fluorescens host cell transformed with a nucleic acid comprising a sequence encoding the recombinant cytokine or chemokine, wherein the nucleic acid does not comprise a secretion signal coding sequence, the process comprising:

(a) growing the transformed P. fluorescens host cell under conditions that allow expression of the recombinant cytokine or chemokine; and

(b) isolating the recombinant cytokine or chemokine expressed;

wherein the recombinant cytokine or chemokine is produced in the cytoplasm of the P. fluorescens host cell as 5-40% total cell protein, wherein the recombinant cytokine or chemokine produced is present in the host cell in a soluble form, and wherein the soluble recombinant cytokine or chemokine is produced at a titre in the P. fluorescens host cell that is about 1.5-fold to about 8-fold higher than the titre of the soluble recombinant cytokine or chemokine produced in an E. coli host cell.

12. The process of claim 11, further comprising substantially purifying the recombinant cytokine or chemokine isolated in step (b).

13. The process of claim 11, wherein the recombinant cytokine or chemokine produced is present in the host cell in an active form.

14. The process of claim 11, wherein the recombinant cytokine or chemokine is a human peptide.

15. The process of claim 11, wherein the recombinant cytokine or chemokine has a mass of between about 1 kD and about 100 kD.

16. The process of claim 15, wherein the recombinant cytokine or chemokine has a mass of between about 1 kD and about 30 kD.

17. The process of claim 11 wherein the cytokine or chemokine is produced as about 10% to 40% total cell protein.

18. The process of claim 11 wherein the cytokine or chemokine is produced as about 15% to 40% total cell protein.

19. The process of claim 11 wherein the cytokine or chemokine is produced as about 20% to 40% total cell protein.

20. The process of claim 11 wherein the cytokine or chemokine is produced as about 30% to 40% total cell protein.