Covalent tethering of functional groups to proteins and substrates therefor

Patent metadata

Field	Value
Publication number	US-10101332-B2
Application number	US-201615238283-A
Country	US
Kind code	B2
Filing date	Aug 16, 2016
Priority date	Jul 30, 2004
Publication date	Oct 16, 2018
Grant date	Oct 16, 2018

Abstract

A mutant hydrolase optionally fused to a protein of interest is provided. The mutant hydrolase is capable of forming a bond with a substrate for the corresponding nonmutant (wild-type) hydrolase which is more stable than the bond formed between the wild-type hydrolase and the substrate and has at least two amino acid substitutions relative to the wild-type hydrolase. Substrates for hydrolases comprising one or more functional groups are also provided, as well as methods of using the mutant hydrolase and the substrates of the invention. Also provided is a fusion protein capable of forming a stable bond with a substrate and cells which express the fusion protein.

First claim

What is claimed is:

1. A composition comprising a dehalogenase substrate of the formula R-linker-A-X, wherein R is peptide or polypeptide, A-X is a substrate for said dehalogenase, X is a halogen, and the linker is a group that separates R and A; wherein R, linker, A, and X are covalently linked.

2. The composition of claim 1 , wherein A is (CH 2 ) n , and n=4-10.

3. The composition of claim 2 , wherein A is (CH 2 ) n , and n=6-10.

4. The composition of claim 1 , wherein the linker is a branched or unbranched carbon chain comprising no more than 30 carbons.

5. The composition of claim 4 , wherein the linker comprises —C(O)NH(CH 2 CH 2 O) y , wherein y=2-8.

6. The composition of claim 1 , wherein X is Cl or Br.

7. The composition of claim 1 , wherein linker-A separates R and X by at least 11 atoms.

8. The composition of claim 1 , wherein the peptide or polypeptide is an antibody, antibody variant, or receptor.

9. The composition of claim 8 , wherein the antibody variant comprises FabFc 2 , Fab, Fv, Fd, F(ab) 2 , an Fv fragment containing only the light or heavy chain variable regions, a Fab or F(ab) 2 fragment containing the variable regions and parts of the constant regions, a single-chain antibody, or CDR-grafted antibodies.

10. The composition of claim 9 , wherein the single-chain antibody is a scFv fragment.

11. The composition of claim 1 , wherein the peptide or polypeptide is a fusion peptide or polypeptide.

12. The composition of claim 11 , wherein the fusion comprises a mutant dehalogenase.

13. A method to label a cell, comprising contacting a cell comprising a mutant dehalogenases with the composition of claim 1 , wherein the mutant dehalogenases comprises at least one amino acid substitution relative to the corresponding wild-type dehalogenases, wherein the at least one amino acid substitution results in the mutant dehalogenase forming a bond with the substrate which is more stable than the bond formed between the corresponding wild-type dehalogenases and the substrate, wherein the at least one amino acid substitution in the mutant dehalogenases is a substitution (i) at an amino acid residue in the corresponding wild-type dehalogenases that is associated with activating a water molecule which cleaves the bond formed between the corresponding wild-type dehalogenases and the substrate, or (ii) at an amino acid residue in the corresponding wild-type dehalogenases that forms an ester intermediate with the substrate.

14. The method of claim 13 , wherein the substrate is a substrate for a Rhodococcus dehalogenase.

15. The method of claim 13 , wherein X is Cl or Br.

16. The method of claim 13 , wherein the linker comprises —C(O)NH(CH 2 CH 2 O) y , wherein y=2-8.

17. A method to detect or determine the presence or amount of a mutant dehalogenases, comprising:
a. contacting a mutant dehalogenase with the composition of claim 1 , wherein the mutant dehalogenase comprises at least one amino acid substitution relative to the corresponding wild-type dehalogenase, wherein the at least one amino acid substitution results in the mutant dehalogenase forming a bond with the substrate which is more stable than the bond formed between the corresponding wild-type dehalogenase and the substrate, wherein the at least one amino acid substitution in the mutant dehalogenase is a substitution (i) at an amino acid residue in the corresponding wild-type dehalogenase that is associated with activating a water molecule which cleaves the bond formed between the corresponding wild-type dehalogenase and the substrate or (ii) at an amino acid residue in the corresponding wild-type dehalogenase that forms an ester intermediate with the substrate; and
b. detecting or determining the presence or amount of the mutant dehalogenase.

18. The method of claim 17 , wherein the substrate is a substrate for a Rhodococcus dehalogenase.

19. The method of claim 17 , wherein X is Cl or Br.

20. The method of claim 17 , wherein the linker comprises —C(O)NH(CH 2 CH 2 O) y , wherein y=2-8.

Assignees

• Promega Corp

Inventors

• Darzins Aldis
• Encell Lance P
• Klaubert Dieter
• Los Georgyi V
• Mcdougall Mark
• Wood Keith V
• Wood Monika G
• Zimprich Chad

Classifications

• C07K16/00

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• C07K2319/61

  containing an enzyme fusion for detection (lacZ, luciferase) · CPC title

• C07K7/06

  having 5 to 11 amino acids · CPC title

• G01N2333/914

  Hydrolases (3) · CPC title

• C07K2319/43

  containing a FLAG-tag · CPC title