Covalent tethering of functional groups to proteins

Patent metadata

Field	Value
Publication number	US-10240184-B2
Application number	US-201615376139-A
Country	US
Kind code	B2
Filing date	Dec 12, 2016
Priority date	Jan 31, 2003
Publication date	Mar 26, 2019
Grant date	Mar 26, 2019

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. 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 a fluorogenic or luminogenic molecule, 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 (CH2)n and n=4-10.

3. The composition of claim 2 , wherein A is (CH2)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(CH2CH2O)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. A method to label a cell, comprising:
(a) contacting a cell comprising a mutant dehalogenases 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 dehalogenases 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;
(b) incubating the cell with the composition, wherein incubation results in the cell being labeled with the fluorogenic or luminogenic molecule; and
(c) detecting a change in fluorescence or luminescence upon said composition binding to said mutant dehalogenase.

9. The method of claim 8 , wherein the substrate is a substrate for a Rhodococcus dehalogenase.

10. The method of claim 8 , wherein X is Cl or Br.

11. The method of claim 8 , wherein the linker comprises —C(O)NH(CH2CH2O)y, wherein y=2-8.

12. 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 a change in fluorescence or luminescence upon said composition binding to said mutant dehalogenase, thereby detecting or determining the presence or amount of the mutant dehalogenase.

13. The method of claim 12 , wherein the substrate is a substrate for a Rhodococcus dehalogenase.

14. The method of claim 12 , wherein X is Cl or Br.

15. The method of claim 12 , wherein the linker comprises —C(O)NH(CH2CH2O)y, wherein y=2-8.

Assignees

• Promega Corp

Inventors

• Wood Keith V
• Los Georgyi V
• Bulleit Robert F
• Klaubert Dieter
• Mcdougall Mark
• Zimprich Chad

Classifications

• G01N33/5005

  involving human or animal cells (immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025) · CPC title

• C12Y308/01005

  Haloalkane dehalogenase (3.8.1.5) · CPC title

• C12Q1/34 Primary

  involving hydrolase · CPC title

• C07D311/78

  Ring systems having three or more relevant rings · CPC title

• G01N33/58

  involving labelled substances (G01N33/53 takes precedence) · CPC title