Methods of producing recombinant heme-binding proteins and uses thereof
The present invention is directed to methods of producing recombinant functional heme-binding proteins with complete heme incorporation and purified preparations of the same. The present invention is further directed to methods of identifying agents that modulate the activity of heme-binding proteins.
1. A method of producing a functional recombinant heme-binding protein comprising:
providing an Escherichia coli host cell that expresses endogenous ferrochelatase protein;
co-expressing in said host cell a non-native recombinant heme-binding protein and a recombinant ferrochelatase protein or a polypeptide thereof which retains iron insertion activity, wherein said host cell expresses both the endogenous ferrochelatase protein and the recombinant ferrochelatase protein or polypeptide thereof; and
culturing said host cell under conditions effective for complete heme incorporation into the recombinantly produced heme-binding protein, thereby producing a functional heme-binding protein that does not contain metal-free porphyrin.
2. The method of claim 1 , wherein said co-expressing is carried out in the presence of one or more heme precursors.
3. The method of claim 2 , wherein the heme precursor is selected from the group consisting of δ-amino levulinic acid, succinyl CoA, glycine, glutamate, glutamate-1-semialdehyde, porphobilinogen, hydroxymethylbilane, and protoporphyrin.
4. The method of claim 1 further comprising:
providing one or more expression vectors encoding the recombinant heme-binding protein and the recombinant ferrochelatase.
5. The method of claim 4 , wherein the one or more expression vectors comprise one expression vector encoding both the recombinant heme-binding protein and the recombinant ferrochelatase.
6. The method of claim 4 , wherein the one or more expression vectors comprise a first expression vector encoding the recombinant heme-binding protein and a second expression vector encoding ferrochelatase.
7. The method of claim 4 , wherein the one or more expression vectors is selected from the group consisting of a bacterial expression vector, fungal expression vector, baculoviral expression vector, plant expression vector, archaeal expression vector, and mammalian expression vector.
8. The method of claim 1 , wherein the heme-binding protein is a mammalian heme-binding protein.
9. The method of claim 1 , wherein the heme-binding protein is a bacterial heme-binding protein.
10. The method of claim 1 , wherein the heme-binding protein is a member of a class of heme-binding proteins selected from the group consisting of globins, cytochromes, bacterioferritins, hydroxylamine oxidoreductases, nitrophorins, peroxidases, cyclooxygenases, catalases, cytochromes P-450s, chloroperoxidases, PAS-domain heme sensors, H-NOX heme sensors, and nitric oxide synthases.
11. A system for producing functional heme-binding proteins comprising:
one or more expression constructs encoding a non-native recombinant heme-binding protein and a recombinant ferrochelatase and
an Escherichia coli host cell that expresses endogenous ferrochelatase protein and contains said one or more expression constructs.
12. The system of claim 11 further comprising:
one or more heme precursors.
13. The system of claim 12 , wherein the one or more heme precursors is selected from the group consisting of δ-amino levulinic acid, succinyl CoA, glycine, glutamate, glutamate-1-semialdehyde, porphobilinogen, hydroxymethylbilane, and protoporphyrin.
14. The system of claim 11 , wherein the one or more expression constructs further comprise a promoter sequence, a nucleic acid encoding a ribosome binding sequence, and a nucleic acid encoding a termination sequence.
15. The system of claim 11 , wherein the one or more expression constructs is selected from the group consisting of a linear DNA construct, a plasmid vector, and a viral vector.
16. The system of claim 11 , wherein the one or more expression constructs comprise one expression construct encoding both the recombinant heme-binding protein and the recombinant ferrochelatase.
17. The system of claim 11 , wherein the one or more expression constructs comprise a first expression construct encoding the recombinant heme-binding protein and a second expression construct encoding the recombinant ferrochelatase.
18. The system of claim 11 , wherein the one or more expression constructs is selected from the group consisting of a bacterial expression vector, fungal expression vector, baculoviral expression vector, plant expression vector, archaeal expression vector, and mammalian expression vector.
19. The system of claim 11 , wherein the heme-binding protein is a mammalian heme-binding protein.
20. The system of claim 11 , wherein the heme-binding protein is a bacterial heme-binding protein.
21. The system of claim 11 , wherein the heme-binding protein is a member of a class of heme-binding proteins selected from the group consisting of cytochromes, bacterioferritins, hydroxylamine oxidoreductases, nitrophorins, peroxidases, cyclooxygenases, catalases, cytochromes P-450s, chloroperoxidases, globins, PAS-domain heme sensors, H-NOX heme sensors, and nitric oxide synthases.
22. The system of claim 11 , wherein the system produces a functional heme-binding protein that does not contain metal-free porphyrin.
23. A method of producing a functional recombinant heme-binding protein comprising:
providing a bacterial or yeast host cell that expresses endogenous ferrochelatase protein;
co-expressing in said host cell (i) a non-native recombinant heme-binding protein that is a member of a class of heme-binding proteins selected from the group consisting of cytochrome P450s, PAS-domain heme sensors, and nitric oxide synthases, and (ii) a recombinant ferrochelatase protein or a polypeptide thereof which retains iron insertion activity, wherein said host cell expresses both the endogenous ferrochelatase protein and the recombinant ferrochelatase protein or polypeptide thereof; and
culturing said host cell under conditions effective for complete heme incorporation into the recombinantly produced heme-binding protein, thereby producing a functional heme-binding protein that does not contain metal-free porphyrin.
24. The method of claim 23 , wherein said co-expressing is carried out in the presence of one or more heme precursors.
25. The method of claim 24 , wherein the heme precursor is selected from the group consisting of δ-amino levulinic acid, succinyl CoA, glycine, glutamate, glutamate-1-semialdehyde, porphobilinogen, hydroxymethylbilane, and protoporphyrin.
26. The method of claim 23 further comprising:
providing one or more expression vectors encoding the recombinant heme-binding protein and the recombinant ferrochelatase.
27. The method of claim 26 , wherein the one or more expression vectors comprise one expression vector encoding both the recombinant heme-binding protein and the recombinant ferrochelatase.
28. The method of claim 26 , wherein the one or more expression vectors comprise a first expression vector encoding the recombinant heme-binding protein and a second expression vector encoding ferrochelatase.
29. The method of claim 26 , wherein the one or more expression vectors is selected from the group consisting of a bacterial expression vector, fungal expression vector, baculoviral expression vector, plant expression vector, archaeal expression vector, and mammalian expression vector.
30. The method of claim 23 , wherein the heme-binding protein is a mammalian heme-binding protein.
31. The method of claim 23 , wherein the heme-binding protein is a bacterial heme-binding protein.
32. A system for producing functional heme-binding proteins comprising:
one or more expression constructs encoding (i) a non-native recombinant heme-binding protein that is a member of a class of heme-binding proteins selected from the group consisting of cytochrome P450s, PAS-domain heme sensors, and nitric oxide synthases, and (ii) a recombinant ferrochelatase and
a bacterial or yeast cell that expresses endogenous ferrochelatase protein and contains said one or more expression constructs.
33. The system of claim 32 further comprising:
one or more heme precursors.
34. The system of claim 32 , wherein the one or more heme precursors is selected from the group consisting of δ-amino levulinic acid, succinyl CoA, glycine, glutamate, glutamate-1-semialdehyde, porphobilinogen, hydroxymethylbilane, and protoporphyrin.
35. The system of claim 32 , wherein the one or more expression constructs further comprise a promoter sequence, a nucleic acid encoding a ribosome binding sequence, and a nucleic acid encoding a termination sequence.
36. The system of claim 32 , wherein the one or more expression constructs is selected from the group consisting of a linear DNA construct, a plasmid vector, and a viral vector.
37. The system of claim 32 , wherein the one or more expression constructs comprise one expression construct encoding both the recombinant heme-binding protein and the recombinant ferrochelatase.
38. The system of claim 32 , wherein the one or more expression constructs comprise a first expression construct encoding the recombinant heme-binding protein and a second expression construct encoding the recombinant ferrochelatase.
39. The system of claim 32 , wherein the one or more expression constructs is selected from the group consisting of a bacterial expression vector, fungal expression vector, baculoviral expression vector, plant expression vector, archaeal expression vector, and mammalian expression vector.
40. The system of claim 32 , wherein the heme-binding protein is a mammalian heme-binding protein.
41. The system of claim 32 , wherein the heme-binding protein is a bacterial heme-binding protein.
42. The system of claim 32 , wherein the system produces a functional heme-binding protein that does not contain metal-free porphyrin.