Aldehyde tags, uses thereof in site-specific protein modification

Patent metadata

Field	Value
Publication number	US-9447390-B2
Application number	US-201414464528-A
Country	US
Kind code	B2
Filing date	Aug 20, 2014
Priority date	Sep 21, 2006
Publication date	Sep 20, 2016
Grant date	Sep 20, 2016

Abstract

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.

First claim

That which is claimed is:

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.

Assignees

• Univ California

Inventors

• Carrico Isaac S
• Carlson Brian L
• Wu Peng
• Bertozzi Carolyn

Classifications

• C12P21/00

  Preparation of peptides or proteins (single cell protein C12N1/00) · CPC title

• C07K2317/24

  containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered · CPC title

• C07K14/35

  from Mycobacteriaceae (F) · CPC title

• C12N9/13 Primary

  transferring sulfur containing groups (2.8) · CPC title

• C07K14/565

  IFN-beta · CPC title