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

• US 20120330001A1
• Filed: 05/22/2012
• Published: 12/27/2012
• Est. Priority Date: 01/31/2003
• Status: Active Grant

• Current Assignee: Promega Corporation
• Original Assignee: Promega Corporation
• Inventors: Darzins, Aldis, Encell, Lance, Johnson, Tonny, Klaubert, Dieter, Los, Georgyi V., McDougall, Mark, Wood, Keith V., Wood, Monika G., Zimprich, Chad
• Granted Patent: US 8,779,221 B2
• Time in Patent Office: Days
• US Class Current: 536/56
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07K 2299/00 Coordinates from 3D structures of peptides, e.g. proteins or enzymes
• CPC Class Codes: C12N 9/0069 acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12N 9/14 Hydrolases (3)
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: C12Q 1/66 involving luciferase
• CPC Class Codes: C12Y 113/12005 Renilla-luciferin 2-monooxygenase (1.13.12.5), i.e. renilla-luciferase
• CPC Class Codes: C12Y 308/01005 Haloalkane dehalogenase (3.8.1.5)

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.

Claims

1. A composition comprising a dehalogenase substrate of the formula R-linker-A-X, wherein R is a solid substrate, A-X is a substrate for said dehalogenase, X is a halogen, and the linker is a group that separates R and A.

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. The composition of claim 1, wherein said 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.