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 modifying a polypeptide, the method comprising:
contacting a non-naturally occurring, recombinant polypeptide comprising a heterologous, converted sulfatase motif generated by reaction with a formylglycine-generating enzyme (FGE) with a reactive partner comprising an aldehyde-reactive group and a moiety of interest, wherein the heterologous, converted sulfatase motif is less than 13 amino acid residues and contains a contiguous sequence of the formula:
X 1 (FGly)X 2 Z 2 X 3 R
(SEQ ID NO: 3)
(I)
wherein
FGly is a 2-formylglycine residue;
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 conjugation of the moiety of interest of the reactive partner to FGly of the polypeptide, thereby producing a modified polypeptide.
2. The method of claim 1 , wherein the heterologous, converted sulfatase motif is positioned in the non-naturally occurring, recombinant polypeptide:
at an internal sequence of the polypeptide;
at a terminal loop, a C-terminus, or an N-terminus of the recombinant polypeptide;
at a solvent-accessible region of the recombinant polypeptide when folded; and/or
at a site of post-translational modification of the recombinant polypeptide that is native or non-native to the amino acid sequence of the recombinant polypeptide.
3. The method of claim 2 , wherein the heterologous sulfatase motif is positioned at an internal sequence of the recombinant polypeptide.
4. The method of claim 2 , wherein the heterologous sulfatase motif is positioned at a terminal loop, a C-terminus, or an N-terminus of the recombinant polypeptide.
5. The method of claim 2 , wherein the heterologous sulfatase motif is positioned on a solvent-accessible region of the recombinant polypeptide when folded.
6. The method of claim 2 , wherein the heterologous sulfatase motif is positioned at a site of post-translational modification of the polypeptide that is native or non-native to the amino acid sequence of the recombinant polypeptide.
7. 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.
8. The method of claim 1 , wherein X 1 is L, M, V, S or T.
9. The method of claim 1 , wherein X 2 and X 3 are each independently S, T, A, V, G or C.
10. The method of claim 9 , wherein X 1 is L, M, V, S or T.
11. The method of claim 10 , wherein the converted sulfatase motif is L(FGly)TPSR (SEQ ID NO: 62).
12. The method of claim 10 , wherein the converted sulfatase motif is selected from M(FGly)TPSR (SEQ ID NO: 63), V(FGly)TPSR (SEQ ID NO: 64), L(FGly)SPSR (SEQ ID NO: 65), L(FGly)APSR (SEQ ID NO: 66), L(FGly)VPSR (SEQ ID NO: 67), and L(FGly)GPSR (SEQ ID NO: 68).
13. 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.
14. The method of claim 1 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc fragment.
15. The method of claim 14 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc polypeptide.
16. The method of claim 15 , wherein the non-naturally occurring, recombinant polypeptide is an antibody.
17. The method of claim 16 , wherein the antibody is an IgG antibody.
18. The method of claim 16 , wherein the antibody is a humanized antibody.
19. The method of claim 1 , wherein the non-naturally occurring, recombinant polypeptide comprises an antigen-binding fragment of an antibody.
20. The method of claim 19 , wherein the non-naturally occurring, recombinant polypeptide comprises a Fab or Fv.
21. The method of claim 1 , wherein the non-naturally occurring, recombinant polypeptide comprises a single chain antibody.
22. The method of claim 1 , wherein the non-naturally occurring, recombinant polypeptide is a blood factor.
23. The method of claim 22 , wherein the blood factor is Factor VIII.
24. The method of claim 1 , wherein the non-naturally occurring, recombinant polypeptide is a fibroblast growth factor.
25. The method of claim 1 , wherein the non-naturally occurring, recombinant polypeptide is a protein vaccine.
26. The method of claim 1 , wherein the non-naturally occurring, recombinant polypeptide is an enzyme.
27. The method of claim 1 , wherein the moiety of interest is a drug.
28. The method of claim 27 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc fragment.
29. The method of claim 27 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc polypeptide.
30. The method of claim 27 , wherein the non-naturally occurring, recombinant polypeptide comprises an antibody.
31. The method of claim 1 , wherein the moiety of interest is a toxin.
32. The method of claim 31 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc fragment.
33. The method of claim 31 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc polypeptide.
34. The method of claim 31 , wherein the non-naturally occurring, recombinant polypeptide comprises an antibody.
35. The method of claim 1 , wherein the moiety of interest is a peptide.
36. The method of claim 35 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc fragment.
37. The method of claim 35 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc polypeptide.
38. The method of claim 35 , wherein the non-naturally occurring, recombinant polypeptide comprises an antibody.
39. The method of claim 1 , wherein the aldehyde-reactive group of the reactive partner comprises an aminooxy.
40. The method of claim 1 , wherein the aldehyde-reactive group of the reactive partner comprises a hydrazide.
41. The method of claim 1 , wherein the aldehyde-reactive group of the reactive partner comprises a thiosemicarbazide.
42. The method of claim 1 , wherein the heterologous, converted sulfatase motif is less than 12 amino acid residues.
43. The method of claim 1 , wherein the heterologous, converted sulfatase motif is less than 11 amino acid residues.
44. The method of claim 1 , wherein the heterologous, converted sulfatase motif is less than 10 amino acid residues.
45. The method of claim 1 , wherein the heterologous, converted sulfatase motif is less than 9 amino acid residues.
46. The method of claim 1 , wherein the heterologous, converted sulfatase motif is less than 8 amino acid residues.
47. The method of claim 1 , wherein the heterologous, converted sulfatase motif is less than 7 amino acid residues.