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 method of producing a protein comprising a formylglycine residue, the method comprising:
culturing a cell comprising:
a formylglycine-generating enzyme (FGE); and
a protein comprising an FGE recognition site,
in a cell culture medium comprising Cu 2+ under cell culture conditions in which the FGE converts a cysteine residue or a serine residue of the FGE recognition site to a formylglycine residue, to produce a protein comprising a formylglycine residue.
2. The method according to claim 1 , wherein the Cu 2+ is present in the cell culture medium at a concentration of from 1 nM to 10 mM.
3. The method according to 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 method according to claim 1 , wherein the FGE is endogenous to the cell.
5. The method according to claim 1 , wherein the cell is genetically modified to express an FGE.
6. The method according to claim 1 , wherein the protein containing an FGE recognition site is endogenous to the cell.
7. The method according to claim 1 , wherein the cell is genetically modified to express the protein containing an FGE recognition site.
8. The method according to claim 1 , wherein the cell is a eukaryotic cell.
9. The method according to claim 8 , wherein the eukaryotic cell is a mammalian cell.
10. The method according to claim 9 , 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.
11. The method according to claim 9 , wherein the mammalian cell is a human cell.
12. The method according to claim 8 , wherein the eukaryotic cell is a yeast cell.
13. The method according to claim 8 , wherein the eukaryotic cell is an insect cell.
14. The method according to claim 1 , wherein the protein is an antibody, an antibody fragment, a ligand, an enzyme, or an antigen.
15. The method according to claim 1 , wherein the protein is an antibody or antibody fragment.
16. The method according to claim 15 , wherein the antibody or antibody fragment is selected from the group consisting of: an IgG or fragment thereof, a Fab, a F(ab′)2, a Fab′, an Fv, an ScFv, a bispecific antibody or fragment thereof, a diabody or fragment thereof, a chimeric antibody or fragment thereof, a monoclonal antibody or fragment thereof, a humanized antibody or fragment thereof, and a fully human antibody or fragment thereof.
17. The method according to claim 15 , wherein the antibody specifically binds to a tumor-associated antigen or a tumor-specific antigen.
18. The method according to claim 17 , wherein the tumor associated antigen or tumor-specific antigen is selected from the group consisting of: HER2, CD19, CD22, CD30, CD33, CD56, CD66/CEACAM5, CD70, CD74, CD79b, CD138, Nectin-4, Mesothelin, Transmembrane glycoprotein NMB (GPNMB), Prostate-Specific Membrane Antigen (PSMA), SLC44A4, CA6, and CA-IX.
19. The method according to claim 1 , wherein the protein is a ligand.
20. The method according to claim 19 , wherein the ligand is a growth factor or a hormone.
21. The method according to claim 1 , further comprising conjugating an agent to the protein comprising the formylglycine residue via an aldehyde moiety of the formylglycine residue.
22. The method according to claim 21 , wherein the agent is a therapeutic agent.
23. The method according to claim 22 , wherein the therapeutic agent is selected from the group consisting of: a cytotoxic agent, an antiproliferative agent, an antineoplastic agent, an antibiotic agent, an antifungal agent, and an antiviral agent.
24. The method according to claim 21 , wherein the agent is an imaging agent.