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

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.

1 . (canceled) 
     
     
         2 . A fusion protein comprising: (a) a mutant dehalogenase having one or more amino acid substitutions relative to a corresponding wild-type dehalogenase, wherein said one or more amino acid substitutions results in the mutant dehalogenase forming a covalent bond with a dehalogenase substrate, and (b) a luciferase. 
     
     
         3 . The fusion protein of  claim 2 , wherein one of the one or more amino acid substitutions is at a residue in the wild-type dehalogenase associated with activating a water molecule which cleaves the covalent bond formed between said wild-type dehalogenase and the substrate. 
     
     
         4 . The fusion protein of  claim 2 , wherein one of the one or more amino acid substitutions is at a residue in the wild-type dehalogenase that forms an ester intermediate with the substrate. 
     
     
         5 . The fusion protein of  claim 2 , wherein the mutant dehalogenase comprises a substitution at an amino acid residue corresponding to residue 106 and/or 272 in a  Rhodococcus  dehalogenase having the amino acid sequence of SEQ ID NO:82. 
     
     
         6 . The fusion protein of  claim 5 , wherein the mutant dehalogenase further comprises a substitution at an amino acid residue corresponding to residue 175, 176 and/or 273 in the  Rhodococcus  dehalogenase. 
     
     
         7 . The fusion protein of  claim 2 , wherein the luciferase is a  Renilla  luciferase. 
     
     
         8 . A polynucleotide encoding the fusion protein of  claim 2 . 
     
     
         9 . A system comprising: (a) a fusion protein of  claim 2 , and (b) a compound of formula (I): R-linker-A-X, wherein R is a functional group, wherein the linker is a branched or unbranched carbon chain comprising from 2 to 30 carbon atoms, which chain optionally includes one or more double or triple bonds, and which chain is optionally substituted with one or more hydroxy or oxo (═O) groups, wherein one or more of the carbon atoms in the chain is optionally replaced with a non-peroxide —O—, —S— or —NH—, wherein the linker-A separates R and X by at least 11 atoms, wherein A is (CH 2 ) n  and n=4-10, wherein A-X is a substrate for the wild-type dehalogenase, and wherein X is a halogen. 
     
     
         10 . The system of  claim 9 , wherein R is selected from the group consisting of a fluorophore, chromophore, luminophore, nucleic acid, a peptide, a polypeptide, a solid support, a radionuclide, a contrast agent, a metal ion chelator, an affinity molecule, a drug, a toxin, a substrate for an enzyme, an inhibitor of an enzyme, a protein ligand, a DNA intercalator, and a crosslinker, and an electron opaque molecule. 
     
     
         11 . The system of  claim 9 , wherein the functional group comprises a protein. 
     
     
         12 . The system of  claim 9 , wherein the functional group comprises an affinity molecule that covalently interacts with an acceptor group. 
     
     
         13 . The system of  claim 9 , wherein X is Cl or Br. 
     
     
         14 . The system of  claim 9 , wherein the linker comprises C(O)NH(CH 2 CH 2 O).