Covalent tethering of functional groups to proteins

Patent metadata

Field	Value
Publication number	US-9540402-B2
Application number	US-201414584834-A
Country	US
Kind code	B2
Filing date	Dec 29, 2014
Priority date	Jan 31, 2003
Publication date	Jan 10, 2017
Grant date	Jan 10, 2017

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 an affinity molecule that forms a covalent bond with an acceptor 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 system comprising the composition of claim 1 and the acceptor molecule.

9. The system of claim 8 , wherein the acceptor molecule is free in solution.

10. The system of claim 8 , wherein the acceptor molecule is bound to a solid support.

11. The system of claim 10 , wherein the solid support is solid support is a surface plasmon resonance chip, a magnetic particle, a membrane, glass, cellulose, self-assembled monolayers, a well of a multi-well plate, an electron conducting surface, or an agarose bead.

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

• C07C217/08

  the oxygen atom of the etherified hydroxy group being further bound to an acyclic carbon atom · CPC title

• C07D209/48

  with oxygen atoms in positions 1 and 3, e.g. phthalimide · CPC title

• Y10S436/80

  Fluorescent dyes, e.g. rhodamine · CPC title

• C07D233/64

  with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine · CPC title