Method of immobilizing a protein or molecule via a mutant dehalogenase that is bound to an immobilized dehalogenase substrate and linked directly or indirectly to the protein or molecule

Patent metadata

Field	Value
Publication number	US-10604745-B2
Application number	US-201815991695-A
Country	US
Kind code	B2
Filing date	May 29, 2018
Priority date	Jan 31, 2003
Publication date	Mar 31, 2020
Grant date	Mar 31, 2020

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 R-linker-A-X-fusion, wherein R is a magnetic bead, wherein A is an alkyl chain consisting of (CH 2 ) n and n=4-10, wherein the linker is a group that separates R and A, wherein X is an ester bond, and wherein the fusion is a fusion of (i) a mutant dehalogenase having at least 85% sequence identity with SEQ ID NO:82, and comprising one or more amino acid substitutions relative to a corresponding wild-type dehalogenase, wherein said one or more amino acid substitutions result in the mutant dehalogenase being capable of forming a covalent ester bond with an alkylhalide substrate, and (ii) protein A or protein G.

2. The composition of claim 1 , wherein the ester bond is between the alkyl chain and a residue of the mutant dehalogenase.

3. The composition of claim 2 , wherein the ester bond is between the alkyl chain and an amino acid of the mutant dehalogenase corresponding to position 106 of SEQ ID NO:82.

4. The composition of claim 1 , wherein the alkyl chain comprises (CH 2 ) n and n is 2-10.

5. The composition of claim 4 , wherein the alkyl chain comprises (CH 2 ) n and n is 6-10.

6. The composition of claim 1 , wherein the linker is a branched or unbranched carbon chain, and wherein: the linker no more than 30 carbons, the linker comprises one or more double or triple bonds, the linker comprises one or more hydroxy or oxo groups, the linker comprises one or more —O—, —S— or —NH— groups.

7. The composition of claim 6 , 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
• Johnson Tonny
• Klaubert Dieter
• Los Georgyi V
• Mcdougall Mark
• Wood Keith V
• Wood Monika G
• Zimprich Chad

Classifications

• C12Q1/66

  involving luciferase · CPC title

• B82Y5/00

  Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery · CPC title

• C12Q1/26

  involving oxidoreductase · CPC title

• C07K2299/00

  Coordinates from 3D structures of peptides, e.g. proteins or enzymes · CPC title

• C12Y308/01005

  Haloalkane dehalogenase (3.8.1.5) · CPC title