Aldehyde tags, uses thereof in site-specific protein modification
View Patent ↗The invention features compositions and methods for site-specific modification of proteins by incorporation of an aldehyde tag. Enzymatic modification at a sulfatase motif of the aldehyde tag through action of a formylglycine generating enzyme (FGE) generates a formylglycine (FGly) residue. The aldehyde moiety of FGly residue can be exploited as a chemical handle for site-specific attachment of a moiety of interest to a polypeptide.
1. A method for producing a 2-formylglycine in a polypeptide, the method comprising:
contacting a non-naturally occurring, recombinant polypeptide comprising a heterologous sulfatase motif with a formylglycine generating enzyme (FGE), wherein the heterologous sulfatase motif is less than 13 amino acid residues and contains a contiguous sequence of the formula
X 1 Z 1 X 2 Z 2 X 3 R (SEQ ID NO: 2) (I)
wherein
Z 1 is cysteine or serine;
Z 2 is a proline or alanine residue;
X 1 , X 2 and X 3 are each independently any amino acid; and
wherein said contacting is under conditions sufficient for conversion of Z 1 to a 2-formylglycine (FGly) residue in the non-naturally occurring, recombinant polypeptide, thereby producing a converted aldehyde tagged polypeptide.
2. The method of claim 1 , wherein the heterologous sulfatase motif of the non-naturally occurring, recombinant polypeptide:
is positioned at a C-terminus of the non-naturally occurring, recombinant polypeptide;
is present at an internal site in a terminal loop of the non-naturally occurring, recombinant polypeptide;
is, where the non-naturally occurring, recombinant polypeptide is a transmembrane protein, present at an internal site within an extracellular loop or an intracellular loop of the transmembrane protein;
is present at an internal site or at an N-terminus and is solvent-accessible when the non-naturally occurring, recombinant polypeptide is folded; and/or
is present at a native or non-native site of post-translational modification which is a glycosylation site.
3. The method of claim 1 , wherein X 1 , X 2 , and X 3 are each independently an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid.
4. The method of claim 1 , wherein X 2 and X 3 are each independently S, T, A, V, G, or C.
5. The method of claim 1 , wherein X 1 is L, M, V, S or T and X 2 and X 3 are each independently S, T, A, V, G or C.
6. The method of claim 1 , wherein X 1 is L, M, V, S or T.
7. The method of claim 6 , wherein the sulfatase motif is LCTPSR (SEQ ID NO: 55).
8. The method of claim 6 , wherein the sulfatase motif is selected from MCTPSR (SEQ ID NO: 56), VCTPSR (SEQ ID NO: 57), LCSPSR (SEQ ID NO: 58), LCAPSR (SEQ ID NO: 59), LCVPSR (SEQ ID NO: 60), and LCGPSR (SEQ ID NO: 61).
9. The method of claim 1 , wherein the recombinant polypeptide is expressed in a cell containing the FGE.
10. The method of claim 1 , wherein the method further comprises:
contacting the converted aldehyde tagged polypeptide with a reactive partner comprising an aldehyde-reactive group and a moiety of interest;
wherein said contacting is under conditions which provide a reaction product having the moiety of interest covalently bound to the FGly residue of the converted aldehyde tagged polypeptide.
11. The method of claim 1 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc fragment.
12. The method of claim 11 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc polypeptide.
13. The method of claim 12 , wherein the non-naturally occurring, recombinant polypeptide is an antibody.
14. The method of claim 13 , wherein the antibody is an IgG antibody.
15. The method of claim 13 , wherein the antibody is a humanized antibody.
16. The method of claim 1 , wherein the non-naturally occurring, recombinant polypeptide comprises an antigen-binding fragment of an antibody.
17. The method of claim 16 , wherein the non-naturally occurring, recombinant polypeptide comprises a Fab or Fv.
18. The method of claim 1 , wherein the non-naturally occurring, recombinant polypeptide comprises a single chain antibody.
19. The method of claim 1 , wherein the non-naturally occurring, recombinant polypeptide is a blood factor.
20. The method of claim 19 , wherein the blood factor is Factor VIII.
21. The method of claim 1 , wherein the non-naturally occurring, recombinant polypeptide is a fibroblast growth factor.
22. The method of claim 1 , wherein the non-naturally occurring, recombinant polypeptide is a protein vaccine.
23. The method of claim 1 , wherein the non-naturally occurring, recombinant polypeptide is an enzyme.
24. The method of claim 1 , wherein the heterologous sulfatase motif is less than 12 amino acid residues.
25. The method of claim 1 , wherein the heterologous sulfatase motif is less than 11 amino acid residues.
26. The method of claim 1 , wherein the heterologous sulfatase motif is less than 10 amino acid residues.
27. The method of claim 1 , wherein the heterologous sulfatase motif is less than 9 amino acid residues.
28. The method of claim 1 , wherein the heterologous sulfatase motif is less than 8 amino acid residues.
29. The method of claim 1 , wherein the heterologous sulfatase motif is less than 7 amino acid residues.
30. The method of claim 1 , wherein said contacting is in vitro.
31. The method of claim 1 , wherein said contacting is performed in a cell-free system.
32. A method for producing a 2-formylglycine in a polypeptide in a cell, the method comprising:
culturing a cell comprising a polynucleotide encoding a non-naturally occurring, recombinant polypeptide comprising a heterologous sulfatase motif and a polynucleotide encoding a formylglycine generating enzyme (FGE);
wherein the heterologous sulfatase motif is less than 13 amino acid residues and comprises a contiguous sequence of the formula:
X 1 Z 1 X 2 Z 2 X 3 R (SEQ ID NO: 2) (I)
wherein
Z 1 is cysteine or serine;
Z 2 is a proline or alanine residue;
X 1 , X 2 and X 3 are each independently any amino acid; and
wherein said culturing is under cellular conditions sufficient for expression of the non-naturally occurring, recombinant polypeptide and the FGE, and sufficient for conversion of a cysteine or serine residue of the heterologous sulfatase motif by the FGE to a 2-formylglycine (FGly) to produce a converted aldehyde tagged polypeptide.
33. The method of claim 32 , wherein the cell is a mammalian cell.
34. The method of claim 32 , wherein the FGE is heterologous to the cell.
35. The method of claim 32 , wherein the heterologous sulfatase motif of the non-naturally occurring, recombinant polypeptide is positioned at a C-terminus of the non-naturally occurring, recombinant polypeptide.
36. The method of claim 32 , wherein the heterologous sulfatase motif of the non-naturally occurring, recombinant polypeptide is present at an internal site in a terminal loop of the non-naturally occurring, recombinant polypeptide.
37. The method of claim 32 , wherein the heterologous sulfatase motif of the non-naturally occurring, recombinant polypeptide is, where the non-naturally occurring, recombinant polypeptide is a transmembrane protein, present at an internal site within an extracellular loop or an intracellular loop of the transmembrane protein.
38. The method of claim 32 , wherein the heterologous sulfatase motif of the non-naturally occurring, recombinant polypeptide is present at an internal site or at an N-terminus and is solvent-accessible when the non-naturally occurring, recombinant polypeptide is folded.
39. The method of claim 32 , wherein the heterologous sulfatase motif of the non-naturally occurring, recombinant polypeptide is present at a native or non-native site of post-translational modification which is a glycosylation site.
40. A method of attaching a moiety of interest to a converted aldehyde tagged polypeptide, the method comprising:
contacting the converted aldehyde tagged polypeptide produced by the method of claim 32 with a reactive partner comprising an aldehyde-reactive group and a moiety of interest;
wherein said contacting is under conditions which provide for a reaction product having the moiety of interest covalently bound to a FGly residue of the converted aldehyde tagged polypeptide.
41. The method of claim 40 , wherein the moiety of interest is a drug.
42. The method of claim 41 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc fragment.
43. The method of claim 41 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc polypeptide.
44. The method of claim 41 , wherein the non-naturally occurring, recombinant polypeptide comprises an antibody.
45. The method of claim 40 , wherein the moiety of interest is a toxin.
46. The method of claim 45 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc fragment.
47. The method of claim 45 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc polypeptide.
48. The method of claim 45 , wherein the non-naturally occurring, recombinant polypeptide comprises an antibody.
49. The method of claim 40 , wherein the moiety of interest is a peptide.
50. The method of claim 49 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc fragment.
51. The method of claim 49 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc polypeptide.
52. The method of claim 49 , wherein the non-naturally occurring, recombinant polypeptide comprises an antibody.