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 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 oxime 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.
2. The modified 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 modified 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 modified non-naturally occurring, recombinant polypeptide of claim 1 , wherein the X 1 , when present, is L, M, V, S or T.
5. The modified 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 modified non-naturally occurring, recombinant polypeptide of claim 1 , wherein the heterologous sulfatase motif is selected from M(FGly′)TPSR, V(FGly′)TPSR, L(FGly′)SPSR, L(FGly′)APSR, L(FGly′)VPSR, and L(FGly′)GPSR.
7. The modified non-naturally occurring, recombinant polypeptide of claim 1 , wherein the heterologous sulfatase motif is L(FGly′)TPSR.
8. The modified non-naturally occurring, recombinant polypeptide of claim 1 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc fragment.
9. The modified non-naturally occurring, recombinant polypeptide of claim 1 , wherein the non-naturally occurring, recombinant polypeptide comprises an Fc polypeptide.
10. The modified non-naturally occurring, recombinant polypeptide of claim 1 , wherein the non-naturally occurring, recombinant polypeptide is an antibody.
11. The modified non-naturally occurring, recombinant polypeptide of claim 10 , wherein the antibody is an IgG antibody.
12. The modified non-naturally occurring, recombinant polypeptide of claim 10 , wherein the antibody is a humanized antibody.
13. The modified non-naturally occurring, recombinant polypeptide of claim 1 , wherein the non-naturally occurring, recombinant polypeptide comprises an antigen-binding fragment of an antibody.
14. The modified non-naturally occurring, recombinant polypeptide of claim 13 , wherein the non-naturally occurring, recombinant polypeptide comprises a Fab or Fv.
15. The modified non-naturally occurring, recombinant polypeptide of claim 1 , wherein the non-naturally occurring, recombinant polypeptide comprises a single chain antibody.
16. The modified non-naturally occurring, recombinant polypeptide of claim 1 , wherein the non-naturally occurring, recombinant polypeptide is a blood factor.
17. The modified non-naturally occurring, recombinant polypeptide of claim 16 , wherein the blood factor is Factor VIII.
18. The modified non-naturally occurring, recombinant polypeptide of claim 1 , wherein the non-naturally occurring, recombinant polypeptide is a fibroblast growth factor.
19. The modified non-naturally occurring, recombinant polypeptide of claim 1 , wherein the non-naturally occurring, recombinant polypeptide is a protein vaccine.
20. The modified non-naturally occurring, recombinant polypeptide of claim 1 , wherein the non-naturally occurring, recombinant polypeptide is an enzyme.
21. The modified non-naturally occurring, recombinant polypeptide 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.
22. The modified non-naturally occurring, recombinant polypeptide of claim 1 , wherein the moiety of interest is a drug.
23. The modified non-naturally occurring, recombinant polypeptide of claim 1 , wherein the moiety of interest is a peptide.