Protein and nucleic acid expression systems
Among the inventions disclosed herein are: nucleic acid expression systems for bacteria having an intracytoplasmic membrane system, including, for example, methanotrophic bacteria; recombinant nucleic acid constructs comprising a pmo promoter operably linked to an expressible nucleic acid; cloning vectors suitable for making recombinant nucleic acid constructs and methods for the production of proteins such as membrane proteins.
1. A methanotrophic bacterium comprising an expression construct encoding a membrane protein that is foreign to the methanotrophic bacterium, said expression construct comprising:
a. an expressible nucleic acid sequence comprising a membrane targeting sequence coding sequence operably linked to a coding sequence for the membrane protein; and
b. a pmo promoter operably linked to the expressible nucleic acid sequence, wherein the pmo promoter comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 7.
2. The methanotrophic bacterium of claim 1 , wherein the membrane-targeting sequence is a membrane-targeting leader sequence, and wherein said membrane targeting sequence coding sequence is positioned 5′ relative to the expressible nucleic acid sequence.
3. The methanotrophic bacterium of claim 2 , wherein the leader sequence is encoded by the nucleic acid sequence of SEQ ID NO: 5.
4. The methanotrophic bacterium of claim 1 , wherein the membrane-targeting sequence is a leader sequence from a particulate methane monooxygenase subunit.
5. The methanotrophic bacterium of claim 4 , wherein the leader sequence comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 9, and SEQ ID NO: 10.
6. The methanotrophic bacterium of claim 1 , further comprising a transcriptional terminator sequence operably linked to the expressible nucleic acid sequence.
7. The methanotrophic bacterium of claim 6 , wherein the transcriptional terminator sequence comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 6, and SEQ ID NO: 8.
8. The methanotrophic bacterium of claim 1 , wherein the expression construct is a vector.
9. The methanotrophic bacterium of claim 8 , wherein the vector is selected from the group consisting of: a plasmid, a cosmid, a suicide vector and a recombinant phage.
10. The methanotrophic bacterium of claim 1 , wherein the expression construct further comprises an origin of replication and/or an antibiotic resistance cassette.
11. The methanotrophic bacterium of claim 1 , wherein the methanotrophic bacterium has an intracytoplasmic membrane system.
12. The methanotrophic bacterium of claim 1 , wherein the methanotrophic bacterium is selected from the group consisting of: a Methylococcus capsulatus , a Methylosinus trichosporium and a Methylocystis sp.
13. A method for producing the membrane protein of claim 1 , comprising culturing the methanotrophic bacterium, under conditions suitable for production of the membrane protein, thereby producing the membrane protein.
14. The method of claim 13 , wherein culturing the cell under conditions suitable for production of the polypeptide comprises exposing the cell to copper(II).
15. The method of claim 14 , wherein the copper(II) is provided at a concentration ranging from about 5 μM to about 50 μM.
16. The method of claim 13 , further comprising: preparing a partially purified polypeptide composition comprising the membrane protein.
17. The method of claim 16 , further comprising: preparing a purified polypeptide composition comprising the membrane protein.
18. A methanotrophic bacterium comprising an expression construct encoding a membrane protein that is foreign to the methanotrophic bacterium, said expression construct comprising:
a. an expressible nucleic acid sequence comprising a coding sequence for a leader sequence for a particulate methane monooxygenase subunit operably linked to a coding sequence for the membrane protein, and wherein the leader sequence targets the membrane protein to an intracytoplasmic membrane; and
b. a viral coat protein promoter, a bacteriophage promoter, or an E. coli lac promoter operably linked to the expressible nucleic acid sequence.
19. The methanotrophic bacterium of claim 18 , wherein said coding sequence for said leader sequence is positioned 5′ relative to the expressible nucleic acid sequence.
20. The methanotrophic bacterium of claim 18 , wherein the leader sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:10.
21. The methanotrophic bacterium of claim 18 , further comprising a transcriptional terminator sequence operably linked to the expressible nucleic acid sequence.
22. The methanotrophic bacterium of claim 21 , wherein the transcriptional terminator sequence comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 6, and SEQ ID NO: 8.
23. The methanotrophic bacterium of claim 18 , wherein the expression construct is a vector.
24. The methanotrophic bacterium of claim 23 , wherein the vector is selected from the group consisting of: a plasmid, a cosmid, a suicide vector and a recombinant phage.
25. The methanotrophic bacterium of claim 18 , wherein the expression construct further comprises an origin of replication and/or an antibiotic resistance cassette.
26. The methanotrophic bacterium of claim 18 , wherein the methanotrophic bacterium has an intracytoplasmic membrane system.
27. The methanotrophic bacterium of claim 18 , wherein the methanotrophic bacterium is selected from the group consisting of: a Methylococcus capsulatus , a Methylosinus trichosporium and a Methylocystis sp.
28. A method for producing the membrane protein of claim 18 , comprising culturing the methanotrophic bacterium, under conditions suitable for production of the membrane protein, thereby producing the membrane protein.
29. The method of claim 28 , further comprising: preparing a partially purified polypeptide composition comprising the membrane protein.
30. The method of claim 29 , further comprising: preparing a purified polypeptide composition comprising the membrane protein.