Method, vector and system for expressing polypeptides
Disclosed herein are methods and systems for expressing a polypeptide in a Rhodospirillum species. The methods include introducing an expression vector having a nucleic acid sequence encoding the polypeptide operably linked to a puc promoter into the Rhodospirillum bacterium and growing the Rhodospirillum bacterium under conditions that allow expression of the polypeptide. Vectors for expressing a membrane polypeptide in a Rhodospirillum species are disclosed. The vectors include a puc promoter and a nucleic acid encoding a membrane polypeptide.
1. A method of expressing a polypeptide in a Rhodospirillum bacterium comprising growing the Rhodospirillum bacterium comprising a nucleic acid sequence encoding the polypeptide operably linked to a promoter selected from the group consisting of SEQ ID NO: 1 and a fragment of SEQ ID NO: 1 under conditions that allow expression of the polypeptide, wherein the Rhodospirillum bacterium is Rhodospirillum rubrum H2.
2. The method of claim 1 , wherein the promoter comprises SEQ ID NO: 2.
3. The method of claim 1 , further comprising isolating the polypeptide.
4. The method of claim 3 , wherein the polypeptide is isolated by enriching for ICM.
5. The method of claim 1 , wherein the polypeptide is a membrane polypeptide.
6. The method of claim 1 , wherein the Rhodospirillum is grown under low oxygen tension.
7. The method of claim 1 , wherein the Rhodospirillum is grown in the presence of an alternative electron acceptor.
8. The method of claim 7 , wherein the alternative electron acceptor is selected from the group consisting of dimethyl sulfoxide, methionine sulfoxide, trimethylamine oxide and combinations thereof.
9. The method of claim 1 , wherein the Rhodospirillum is grown to late log phase.
10. The method of claim 1 , wherein the Rhodospirillum is grown semi-aerobically.
11. The method of claim 1 , wherein the Rhodospirillum has reduced expression of a protein natively associated with the intracytoplasmic membrane as compared to the corresponding wild-type Rhodospirillum.
12. The method of claim 11 , wherein the Rhodospirillum species has reduced expression of at least one of RC-H, RC-L, RC-M, LH-α, or LH-β.
13. The method of claim 11 , wherein the Rhodospirillum species comprises a mutation in at least one of RC-H, RC-L, RC-M, LH-α, or LH-β.
14. The method of claim 1 , wherein the nucleic acid sequence encoding the polypeptide is part of a vector.
15. A method of producing a Rhodospirillum bacterium capable of expressing a polypeptide comprising:
introducing a vector comprising a nucleic acid sequence encoding the polypeptide operably linked to a promoter selected from the group consisting of SEQ ID NO: 1 and a fragment of SEQ ID NO: 1 into the Rhodospirillum bacterium, wherein the Rhodospirillum bacterium is Rhodospirillum rubrum H2.
16. The method of claim 15 , wherein the promoter comprises SEQ ID NO: 2.
17. The method of claim 15 , wherein the polypeptide is a membrane polypeptide.
18. The method of claim 15 , wherein the Rhodospirillum has reduced ability to express a protein natively associated with the intracytoplasmic membrane as compared to the corresponding wild-type Rhodospirillum.
19. A nucleic acid encoding a membrane polypeptide operably connected to a promoter selected from the group consisting of SEQ ID NO: 1 and a fragment of SEQ ID NO: 1, wherein the membrane polypeptide is not natively associated with the promoter.
20. The nucleic acid of claim 19 , wherein the promoter comprises SEQ ID NO:2.
21. The nucleic acid of claim 19 , further comprising a terminator fragment.
22. The nucleic acid of claim 19 , wherein the nucleic acid further comprises a sequence encoding an affinity tag.
23. The nucleic acid of claim 19 , further comprising a selectable or screenable marker for selection in a Rhodospirillum.
24. The nucleic acid of claim 19 , wherein a vector comprises the nucleic acid.
25. A Rhodospirillum comprising the nucleic acid of claim 19 , wherein the Rhodospirillum is Rhodospirillum rubrum H2.
26. A system for expressing a polypeptide in a Rhodospirillum comprising the Rhodospirillum and a vector comprising a promoter selected from the group consisting of SEQ ID NO: 1 and a fragment of SEQ ID NO: 1, wherein the Rhodospirillum bacterium is Rhodospirillum rubrum H2.
27. The system of claim 26 , wherein the promoter comprises SEQ ID NO:2.
28. The system of claim 26 , wherein the polypeptide is a membrane polypeptide.
29. The system of claim 26 , wherein the Rhodospirillum has reduced ability to express a protein natively associated with the intracytoplasmic membrane as compared to the corresponding wild-type Rhodospirillum.
30. A Rhodospirillum bacterium comprising Rhodospirillum rubrum H2.