Methods for G-CSF production in a
View Patent ↗The present invention relates to the field of recombinant protein production in bacterial hosts. It further relates to expression of soluble, active recombinant protein by using secretion signals to direct the protein to the periplasmic space of a bacterial cell. In particular, the present invention relates to a production process for obtaining soluble hG-CSF protein from a bacterial host.
1. A method comprising producing a soluble G-CSF protein in a Pseudomonad host cell, wherein at least 99% of the G-CSF protein comprises an N-terminal methionine, wherein the Pseudomonad host cell has a mutation in at least one gene encoding at least one protease, wherein the mutation results in the inactivation of the at least one protease, and wherein the at least one protease inactivated is PrtB; both PrtB and AprA; both the serine peptidase encoded by SEQ ID NO: 29 and the S41 protease encoded by SEQ ID NO: 30; an HtpX protease encoded by SEQ ID NO: 31; or both the serine peptidase encoded by SEQ ID NO: 29 and the Lon protease encoded by SEQ ID NO: 32.
2. The method of claim 1 , wherein the producing comprises expressing the G-CSF protein from an expression construct.
3. The method of claim 2 , wherein the expression construct is in a plasmid.
4. The method of claim 2 , wherein the expression construct comprises a sequence encoding the G-CSF protein fused to a secretion leader.
5. The method of claim 4 , wherein the secretion signal directs transfer of the G-CSF protein to the periplasm of the Pseudomonad host cell.
6. The method of claim 4 , wherein the secretion signal is cleaved from the G-CSF protein in the Pseudomonad host cell.
7. The method of claim 4 , wherein the secretion leader protein sequence comprises any one of SEQ ID NOs: 8-26.
8. The method of claim 1 , wherein the mutation that results in the inactivation of the at least one protease is a complete deletion.
9. The method of claim 1 , wherein the Pseudomonad host cell is a Pseudomonas host cell.
10. The method of claim 9 , wherein the Pseudomonas host cell is a Pseudomonas fluorescens host cell.
11. The method of claim 1 , wherein the G-CSF protein is human G-CSF protein.
12. The method of claim 1 , further comprising determining the yield of the G-CSF protein, wherein the yield of the G-CSF protein is about 0.1 g/L to about 10 g/L.
13. The method of claim 1 , further comprising determining the activity of the G-CSF protein.
14. The method of claim 13 , wherein said activity is determined by binding recombinant G-CSF receptor.
15. A method comprising producing a soluble G-CSF protein in a Pseudomonad host cell, wherein at least 99% of the G-CSF protein comprises an N-terminal methionine, wherein refolding the Met-G-CSF protein is not required, and wherein the yield of soluble Met-G-CSF protein is about 0.1 g/L to about 10 g/L.
16. The method of claim 15 , wherein the producing comprises expressing the G-CSF protein from an expression construct.
17. The method of claim 16 , wherein the expression construct is a plasmid.
18. The method of claim 16 , wherein the expression construct comprises a sequence encoding G-CSF protein fused to a secretion leader.
19. The method of claim 18 , wherein the secretion leader directs transfer of the G-CSF protein to the periplasm of the Pseudomonad host cell.
20. The method of claim 18 , wherein the secretion leader is cleaved from the G-CSF protein in the Pseudomonad host cell.
21. The method of claim 18 , wherein the secretion leader comprises any one of SEQ ID NOs: 8-26.
22. The method of claim 15 , wherein the Pseudomonad host cell is a Pseudomonas host cell.
23. The method of claim 22 , wherein the Pseudomonas host cell is a Pseudomonas fluorescens host cell.
24. The method of claim 15 , wherein the G-CSF protein is human G-CSF protein.
25. The method of claim 15 , further comprising determining the activity of the G-CSF protein.
26. The method of claim 25 , wherein the activity is determined by binding recombinant G-CSF receptor.