Aldehyde Tags, Uses Thereof in Site-Specific Protein Modification

• US 20080187956A1
• Filed: 09/18/2007
• Published: 08/07/2008
• Est. Priority Date: 09/21/2006
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Wu, Peng, Bertozzi, Carolyn, Carlson, Brian L., Carrico, Isaac S.
• Granted Patent: US 7,985,783 B2
• Time in Patent Office: Days
• US Class Current: 435/68.1
• CPC Class Codes: A61K 38/1825 Fibroblast growth factor [FGF]
• CPC Class Codes: A61K 38/215 IFN-beta
• CPC Class Codes: A61K 38/37 Factors VIII
• CPC Class Codes: A61K 38/45 Transferases (2)
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: C07K 1/006 of peptides containing derivatised side chain amino acids
• CPC Class Codes: C07K 1/1072 by covalent attachment of residues or functional groups
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 14/35 from Mycobacteriaceae (F)
• CPC Class Codes: C07K 14/565 IFN-beta
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/46 Hybrid immunoglobulins hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/40 characterized by post-translational modification
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C12N 9/13 transferring sulfur containing groups (2.8)
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12P 21/00 : Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: C12P 21/005 : Glycopeptides, glycoproteins
• CPC Class Codes: C12Y 208/02 : Sulfotransferases (2.8.2)

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.

Claims

1. A method for modifying a polypeptide, the method comprising:

contacting a polypeptide comprising a converted sulfatase motif with a reactive partner comprising a moiety of interest, wherein the converted sulfatase motif comprises;

2. The method of claim 1, wherein the sulfatase motif is a heterologous sulfatase motif.

3. The method of claim 1, wherein the polypeptide comprises an FGly residue positioned:

at an internal sequence of the polypeptide;

at a terminal loop, a C-terminus, or an N-terminus of the polypeptide;

on a solvent-accessible region of the polypeptide when folded; and

/or

at a site of post-translational modification of the polypeptide that is native or non-native to the amino acid sequence of the polypeptide.

4. The method of claim 1, wherein X₁, when present, X₂, and X₃ are each independently an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid.

5. The method of claim 1, wherein the X₁, when present, is L, M, V, S or T.

6. The method of claim 1, wherein X₂ and X₃ are each independently S, T, A, V, G or C.

7. A method for producing a formylglycine in a polypeptide, the method comprising:

contacting a polypeptide comprising a heterologous sulfatase motif with a formylglycine generating enzyme (FGE), wherein the heterologous sulfatase motif of the formula

8. The method of claim 7, wherein the heterologous sulfatase motif of the polypeptide

is less than 16 amino acid residues in length and is positioned at a C-terminus of the polypeptide;

is present at an internal site in a terminal loop of the polypeptide;

is, where the polypeptide is a transmembrane protein, present at an internal site within an extracellular loop or an intracellular loop of the transmembrane protein;

is present at an internal site or at the N-terminus and is solvent-accessible when the polypeptide is folded; and

/or

is present at a native or non-native site of post-translational modification which is a glycosylation site.

9. The method of claim 7, wherein X₁, when present, X₂, and X₃ are each independently an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid.

10. The method of claim 7, wherein X₁, when present, is L, M, V, S or T.

11. The method of claim 7, wherein X₂ and X₃ are each independently S, T, A, V, G, or C.

12. The method of claim 7, wherein the polypeptide is expressed in a cell containing the FGE.

13. The method of claim 7, wherein the method further comprises:

contacting the converted aldehyde tagged polypeptide with a reactive partner comprising a moiety of interest;

wherein said contacting is under conditions to provide for production of a reaction product of a modified aldehyde tagged polypeptide having the moiety of interest covalently bound to the FGly residue of the heterologous sulfatase motif.

14. A polypeptide comprising a heterologous sulfatase motif having a formylglycine residue, wherein the heterologous sulfatase motif comprises

15. The polypeptide of claim 14, wherein the heterologous sulfatase motif

comprises less than 16 amino acid residues and is positioned at a C-terminus of the polypeptide;

is present in a terminal loop of the polypeptide;

is, when the polypeptides 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 polypeptide, and is solvent-accessible when the polypeptide is folded; and

/or

is present at a site of post-translational modification.

16. The method of claim 14, wherein X₁, when present, X₂, and X₃ are each independently an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid.

17. The polypeptide of claim 14, wherein the X₁, when present, is L, M, V, S or T.

18. The method of claim 14, wherein X₂ and X₃ are each independently S, T, A, V, G, or C.

19. A nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide of claim 14.

20. A modified polypeptide comprising a formylglycine residue covalently attached to a moiety of interest, wherein the polypeptide comprises a modified sulfatase motif of the formula:

21. The modified polypeptide of claim 20, wherein the moiety is a water-soluble polymer, a detectable label, a drug, or a moiety for immobilization of the polypeptide in a membrane or on a surface.

22. The modified polypeptide of claim 20, 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.

23. A recombinant nucleic acid comprising:

an expression cassette comprising;

a first nucleic acid comprising an aldehyde tag-encoding sequence; and

a restriction site positioned 5′

or 3′

of the aldehyde tag-encoding sequence, which restriction site provides for insertion of a second nucleic acid encoding a polypeptide of interest; and

a promoter operably linked to the expression cassette to provide for expression of an aldehyde tagged-polypeptide produced by insertion of the second nucleic acid encoding the polypeptide of interest into the restriction site.

24. A reaction mixture comprising:

an isolated Mycobacterium tuberculosis formylglycine generating enzyme (FGE); and

a polypeptide comprising a heterologous sulfatase motif of the formula;