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 non-naturally occurring, recombinant polypeptide comprising a heterologous sulfatase motif having a 2-formylglycine residue, wherein the heterologous sulfatase motif is less than 13 amino acid residues and contain a contiguous sequence of the formula:
X 1 (FGly)X 2 Z 2 X 3 R
where
FGly is a 2-formylglycine residue;
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 aldehyde tagged polypeptide, X 1 is present; and
X 2 and X 3 are each independently any amino acid,
wherein the heterologous sulfatase motif does not include a threonine-glycine-arginine (TGR) motif C-terminal to the arginine residue of the heterologous sulfatase motif.
2. The non-naturally occurring, recombinant polypeptide of claim 1 , wherein the heterologous sulfatase motif
is positioned at a C-terminus of the non-naturally occurring, recombinant polypeptide;
is present in a terminal loop of the non-naturally occurring, recombinant polypeptide;
is, when the non-naturally occurring, recombinant polypeptide is a transmembrane protein, present at an internal site within an extracellular loop or an intracellular loop;
is present at an internal site or at the N-terminus of the non-naturally occurring, recombinant polypeptide, and is solvent-accessible when the polypeptide is folded; and/or
is present at a site of post-translational modification.
3. The non-naturally occurring, recombinant polypeptide 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.
4. The non-naturally occurring, recombinant polypeptide of claim 1 , wherein the X 1 , when present, is L, M, V, S or T.
5. The method non-naturally occurring, recombinant polypeptide of claim 1 , wherein X 2 and X 3 are each independently S, T, A, V, G, or C.
6. The non-naturally occurring, recombinant polypeptide of claim 1 , wherein the heterologous sulfatase motif is L(FGly)TPSR.
7. 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 contain a contiguous sequence of the formula:
X 1 (FGly′)X 2 Z 2 X 3 R
where
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, and
wherein the heterologous sulfatase motif does not include a threonine-glycine-arginine (TGR) motif C-terminal to the arginine residue of the heterologous sulfatase motif.
8. The modified non-naturally occurring, recombinant polypeptide of claim 7 , wherein the moiety of interest is a water-soluble polymer, a detectable label, a drug, a toxin, a peptide, or an immobilization tag.
9. The modified non-naturally occurring, recombinant polypeptide of claim 7 , wherein the modified sulfatase motif is positioned in the modified polypeptide at a site of post-translational modification of that is native or non-native to the parent of the modified polypeptide.
10. The modified non-naturally occurring, recombinant polypeptide of claim 7 , 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.
11. The modified non-naturally occurring, recombinant polypeptide of claim 7 , wherein the moiety of interest is a drug.
12. The modified non-naturally occurring, recombinant polypeptide of claim 7 , wherein the moiety of interest is a peptide.
13. A non-naturally occurring, recombinant polypeptide comprising a heterologous sulfatase motif having a 2-formylglycine residue, wherein the heterologous sulfatase motif is less than 9 amino acid residues and contains a contiguous sequence of the formula:
X 1 (FGly)X 2 Z 2 X 3 R
where
FGly is a 2-formylglycine residue;
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 aldehyde tagged polypeptide, X 1 is present; and
X 2 and X 3 are each independently any amino acid.
14. The non-naturally occurring, recombinant polypeptide of claim 13 , wherein the X 1 , when present, is L, M, V, S or T.
15. The non-naturally occurring, recombinant polypeptide of claim 13 , wherein X 2 and X 3 are each independently S, T, A, V, G, or C.
16. The non-naturally occurring, recombinant polypeptide of claim 13 , wherein the heterologous sulfatase motif is L(FGly)TPSR.
17. The non-naturally occurring, recombinant polypeptide of claim 13 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc fragment.
18. The non-naturally occurring, recombinant polypeptide of claim 13 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc polypeptide.
19. The non-naturally occurring, recombinant polypeptide of claim 13 , wherein the non-naturally occurring, recombinant polypeptide is an antibody.
20. The non-naturally occurring, recombinant polypeptide of claim 19 , wherein the antibody is an IgG antibody.
21. The non-naturally occurring, recombinant polypeptide of claim 19 , wherein the antibody is a humanized antibody.
22. The non-naturally occurring, recombinant polypeptide of claim 13 , wherein the non-naturally occurring, recombinant polypeptide comprises an antigen-binding fragment of an antibody.
23. The non-naturally occurring, recombinant polypeptide of claim 22 , wherein the non-naturally occurring, recombinant polypeptide comprises a Fab or Fv.
24. The non-naturally occurring, recombinant polypeptide of claim 13 , wherein the non-naturally occurring, recombinant polypeptide comprises a single chain antibody.
25. The non-naturally occurring, recombinant polypeptide of claim 13 , wherein the non-naturally occurring, recombinant polypeptide is a blood factor.
26. The non-naturally occurring, recombinant polypeptide of claim 25 , wherein the blood factor is Factor VIII.
27. The non-naturally occurring, recombinant polypeptide of claim 13 , wherein the non-naturally occurring, recombinant polypeptide is a fibroblast growth factor.
28. The non-naturally occurring, recombinant polypeptide of claim 13 , wherein the non-naturally occurring, recombinant polypeptide is a protein vaccine.
29. The non-naturally occurring, recombinant polypeptide of claim 13 , wherein the non-naturally occurring, recombinant polypeptide is an enzyme.
30. 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 9 amino acid residues and contains a contiguous sequence of the formula:
X 1 (FGly′)X 2 Z 2 X 3 R
where
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.
31. The modified non-naturally occurring, recombinant polypeptide of claim 30 , wherein the X 1 , when present, is L, M, V, S or T.
32. The modified non-naturally occurring, recombinant polypeptide of claim 30 , wherein X 2 and X 3 are each independently S, T, A, V, G, or C.
33. The modified non-naturally occurring, recombinant polypeptide of claim 30 , wherein the heterologous sulfatase motif is L(FGly′)TPSR.
34. The modified non-naturally occurring, recombinant polypeptide of claim 30 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc fragment.
35. The modified non-naturally occurring, recombinant polypeptide of claim 30 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc polypeptide.
36. The modified non-naturally occurring, recombinant polypeptide of claim 30 , wherein the non-naturally occurring, recombinant polypeptide is an antibody.
37. The modified non-naturally occurring, recombinant polypeptide of claim 36 , wherein the antibody is an IgG antibody.
38. The modified non-naturally occurring, recombinant polypeptide of claim 36 , wherein the antibody is a humanized antibody.
39. The modified non-naturally occurring, recombinant polypeptide of claim 30 , wherein the non-naturally occurring, recombinant polypeptide comprises an antigen-binding fragment of an antibody.
40. The modified non-naturally occurring, recombinant polypeptide of claim 39 , wherein the non-naturally occurring, recombinant polypeptide comprises a Fab or Fv.
41. The modified non-naturally occurring, recombinant polypeptide of claim 30 , wherein the non-naturally occurring, recombinant polypeptide comprises a single chain antibody.
42. The modified non-naturally occurring, recombinant polypeptide of claim 30 , wherein the non-naturally occurring, recombinant polypeptide is a blood factor.
43. The modified non-naturally occurring, recombinant polypeptide of claim 42 , wherein the blood factor is Factor VIII.
44. The modified non-naturally occurring, recombinant polypeptide of claim 30 , wherein the non-naturally occurring, recombinant polypeptide is a fibroblast growth factor.
45. The modified non-naturally occurring, recombinant polypeptide of claim 30 , wherein the non-naturally occurring, recombinant polypeptide is a protein vaccine.
46. The modified non-naturally occurring, recombinant polypeptide of claim 30 , wherein the non-naturally occurring, recombinant polypeptide is an enzyme.
47. The modified non-naturally occurring, recombinant polypeptide of claim 30 , wherein the moiety of interest is a drug.
48. The modified non-naturally occurring, recombinant polypeptide of claim 30 , wherein the moiety of interest is a peptide.
49. 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 9 amino acid residues and contains a contiguous sequence of the formula:
X 1 (FGly′)X 2 Z 2 X 3 R
where
FGly′ is the 2-formylglycine residue covalently attached to the moiety of interest through a hydrazone, oxime or semicarbazone linkage;
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.