Activated formylglycine-generating enzymes and methods of producing and using the same
The present disclosure provides activated formylglycine-generating enzymes (FGE), methods of producing activated FGE, and their use in methods of producing a protein comprising a formylglycine (FGly) residue. The methods of producing activated FGE, as well as methods of use of activated FGE in producing FGly-containing proteins, include both cell-based and cell-free methods. Compositions and kits that find use, e.g., in practicing the methods of the present disclosure are also provided.
1. A non-naturally occurring composition comprising:
a cell culture medium comprising Cu 2+ ; and
a cell present in the cell culture medium, wherein the cell expresses formylglycine-generating enzyme (FGE).
2. The composition of claim 1 , wherein the Cu 2+ is present in the cell culture medium at a concentration of from 0.1 μM to 10 mM.
3. The composition of claim 2 , wherein the Cu 2+ is present in the cell culture medium at a concentration of from 1 μM to 1 mM.
4. The composition of claim 1 , wherein the FGE is endogenous to the cell.
5. The composition of claim 1 , wherein the cell is genetically modified to express an FGE.
6. The composition of claim 1 , wherein the cell is genetically modified to express a protein containing an FGE recognition site.
7. The composition of claim 1 , wherein the cell is a eukaryotic cell.
8. The composition according to claim 7 , wherein the eukaryotic cell is a mammalian cell.
9. The composition of claim 8 , wherein the mammalian cell is selected from the group consisting of: a CHO cell, a HEK cell, a BHK cell, a COS cell, a Vero cell, a Hela cell, an NIH 3T3 cell, a Huh-7 cell, a PC12 cell, a RAT1 cell, a mouse L cell, an HLHepG2 cell, an NSO cell, a C127 cell, a hybridoma cell, a PerC6 cell, a CAP cell, and a Sp-2/0 cell.
10. The composition according to claim 7 , wherein the eukaryotic cell is a yeast cell.
11. The composition according to claim 7 , wherein the eukaryotic cell is an insect cell.
12. The composition of claim 1 , wherein the cell is a prokaryotic cell.
13. A method comprising:
culturing a cell that comprises a nucleic acid encoding a formylglycine-generating enzyme (FGE) in a non-naturally occurring composition comprising a cell culture medium that comprises Cu 2+ , wherein the culturing is under conditions in which the FGE is expressed in the cell.
14. The method according to claim 13 , wherein the Cu 2+ is present in the cell culture medium at a concentration of from 0.1 μM to 10 mM.
15. The method according to claim 13 , wherein the Cu 2+ is present in the cell culture medium at a concentration of from 1 μM to 1 mM.
16. The method according to claim 13 , wherein the FGE is endogenous to the cell.
17. The method according to claim 13 , wherein the cell is genetically modified to express an FGE.
18. The method according to claim 13 , wherein the cell is genetically modified to express a protein containing an FGE recognition site.
19. The method according to claim 13 , wherein the cell is a eukaryotic cell.
20. The method according to claim 19 , wherein the eukaryotic cell is a mammalian cell.
21. The method according to claim 20 , wherein the mammalian cell is selected from the group consisting of: a CHO cell, a HEK cell, a BHK cell, a COS cell, a Vero cell, a Hela cell, an NIH 3T3 cell, a Huh-7 cell, a PC12 cell, a RAT1 cell, a mouse L cell, an HLHepG2 cell, an NSO cell, a C127 cell, a hybridoma cell, a PerC6 cell, a CAP cell, and a Sp-2/0 cell.
22. The method according to claim 19 , wherein the eukaryotic cell is a yeast cell.
23. The method according to claim 19 , wherein the eukaryotic cell is an insect cell.
24. The method according to claim 13 , wherein the cell is a prokaryotic cell.
25. A method of producing an activated formylglycine-generating enzyme (FGE), comprising treating an FGE with Cu 2+ to produce an activated FGE.
26. The method according to claim 25 , wherein treating the FGE with Cu 2+ comprises culturing a cell that comprises a nucleic acid encoding the FGE in a cell culture medium that comprises Cu 2+ , wherein the culturing is under conditions in which the FGE is expressed in the cell.
27. The method according to claim 26 , wherein the Cu 2+ is present in the cell culture medium at a concentration of from 0.1 μM to 10 mM.
28. The method according to claim 26 , wherein the Cu 2+ is present in the cell culture medium at a concentration of from 1 μM to 1 mM.
29. The method according to claim 26 , wherein the FGE is endogenous to the cell.
30. The method according to claim 26 , wherein the cell is genetically modified to express an FGE.
31. The method according to claim 26 , wherein the cell is a eukaryotic cell.
32. The method according to claim 31 , wherein the eukaryotic cell is a mammalian cell.
33. The method according to claim 32 , wherein the mammalian cell is selected from the group consisting of: a CHO cell, a HEK cell, a BHK cell, a COS cell, a Vero cell, a Hela cell, an NIH 3T3 cell, a Huh-7 cell, a PC12 cell, a RAT1 cell, a mouse L cell, an HLHepG2 cell, an NSO cell, a C127 cell, a hybridoma cell, a PerC6 cell, a CAP cell, and a Sp-2/0 cell.
34. The method according to claim 31 , wherein the eukaryotic cell is a yeast cell.
35. The method according to claim 31 , wherein the eukaryotic cell is an insect cell.
36. The method according to claim 26 , wherein the cell is a prokaryotic cell.
37. The method according to claim 26 , further comprising purifying the FGE from the cell.