Expression of mammalian proteins in
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.
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. 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.
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.