Covalent tethering of functional groups to proteins
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.
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.