Aldehyde tags, uses thereof in site-specific protein modification
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 comprising:
administering a polypeptide to a subject, wherein the polypeptide is a modified non-naturally occurring, recombinant polypeptide comprising a heterologous sulfatase motif having a 2-formylglycine residue covalently attached to a moiety of interest, wherein the heterologous 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: 97)
wherein
FGly′ is the 2-formylglycine residue covalently attached to the moiety of interest;
Z 2 is a proline or alanine residue;
X 1 is present or absent and, when present, is any amino acid, with the proviso that when the heterologous sulfatase motif is at an N-terminus of the polypeptide, X 1 is present; and
X 2 and X 3 are each independently any amino acid.
2. The method of claim 1 , wherein
the heterologous sulfatase motif is positioned at a C-terminus of the modified non-naturally occurring, recombinant polypeptide;
the modified non-naturally occurring, recombinant polypeptide comprises a terminal loop and the heterologous sulfatase motif is present in the terminal loop of the modified non-naturally occurring, recombinant polypeptide;
the heterologous sulfatase motif is, when the modified non-naturally occurring, recombinant polypeptide is a transmembrane protein, present at an internal site within an extracellular loop or an intracellular loop;
the heterologous sulfatase motif is present at an internal site or at the N-terminus of the modified non-naturally occurring, recombinant polypeptide, and is solvent-accessible when the polypeptide is folded; or
the heterologous sulfatase motif is present at a site of post-translational modification.
3. The method of claim 1 , wherein the heterologous sulfatase motif is positioned in the modified non-naturally occurring, recombinant polypeptide at a site of post-translational modification of that is native or non-native to the parent of the modified non-naturally occurring, recombinant polypeptide.
4. The method of claim 1 , wherein the modified non-naturally occurring, recombinant polypeptide is selected from the group consisting of an Fc fragment, an antibody, an antigen-binding fragment of an antibody, a blood factor, a fibroblast growth factor, a protein vaccine, and an enzyme.
5. The method of claim 1 , wherein the modified non-naturally occurring, recombinant polypeptide comprises an Fc fragment.
6. The method of claim 1 , wherein the modified non-naturally occurring, recombinant polypeptide comprises an Fc polypeptide.
7. The method of claim 1 , wherein the modified non-naturally occurring, recombinant polypeptide is an antibody.
8. The method of claim 7 , wherein the antibody is an IgG antibody.
9. The method of claim 7 , wherein the antibody is a humanized antibody.
10. The method of claim 1 , wherein the modified non-naturally occurring, recombinant polypeptide comprises an antigen-binding fragment of an antibody.
11. The method of claim 10 , wherein the modified non-naturally occurring, recombinant polypeptide comprises a Fab or Fv.
12. The method of claim 1 , wherein the modified non-naturally occurring, recombinant polypeptide comprises a single chain antibody.
13. The method of claim 1 , wherein the modified non-naturally occurring, recombinant polypeptide is a blood factor.
14. The method of claim 13 , wherein the blood factor is Factor VIII.
15. The method of claim 1 , wherein the modified non-naturally occurring, recombinant polypeptide is a fibroblast growth factor.
16. The method of claim 1 , wherein the modified non-naturally occurring, recombinant polypeptide is a protein vaccine.
17. The method of claim 1 , wherein the modified non-naturally occurring, recombinant polypeptide is an enzyme.
18. The method of claim 1 , wherein the moiety of interest is a water-soluble polymer, a detectable label, a drug, a toxin, a peptide, or an immobilization tag.
19. The method of claim 1 , wherein the moiety of interest is a drug.
20. The method of claim 1 , wherein the moiety of interest is a peptide.
21. The method of claim 1 , wherein X 1 , when present, X 2 , and X 3 are each independently an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid.
22. The method of claim 1 , wherein the X 1 , when present, is L, M, V, S or T.
23. The method of claim 1 , wherein X 2 and X 3 are each independently S, T, A, V, or C.
24. The method of claim 1 , wherein the heterologous sulfatase motif is selected from M(FGly′)TPSR (SEQ ID NO: 123), V(FGly′)TPSR (SEQ ID NO: 124), L(FGly′)SPSR (SEQ ID NO: 125), L(FGly′)APSR (SEQ ID NO: 126), L(FGly′)VPSR (SEQ ID NO: 127), and L(FGly′)GPSR (SEQ ID NO: 128).
25. The method of claim 1 , wherein the heterologous sulfatase motif is L(FGly′)TPSR (SEQ ID NO: 129).
26. The method of claim 1 , wherein the heterologous sulfatase motif is less than 12 amino acid residues and greater than or equal to 5 amino acid residues.
27. The method of claim 1 , wherein the heterologous sulfatase motif is less than 11 amino acid residues and greater than or equal to 5 amino acid residues.
28. The method of claim 1 , wherein the heterologous sulfatase motif is less than 10 amino acid residues and greater than or equal to 5 amino acid residues.
29. The method of claim 1 , wherein the heterologous sulfatase motif is less than 9 amino acid residues and greater than or equal to 5 amino acid residues.
30. The method of claim 1 , wherein the heterologous sulfatase motif is less than 8 amino acid residues and greater than or equal to 5 amino acid residues.
31. The method of claim 1 , wherein the heterologous sulfatase motif is less than 7 amino acid residues and greater than or equal to 5 amino acid residues.