Mutant luciferase

Abstract

An isolated recombinant luciferase having luciferase activity. The recombinant luciferase has an amino acid sequence which differs from the wild-type luciferase from Photinus pyralis, Luciola mingrelica, Luciola cruciata, Luciola lateralis, Hotaria parvula, Pyrophorus plagiophthalamus, Lampyris noctiluca, Pyrocoelia miyako or Photinus pennsylvanica. In the sequence of the recombinant luciferase, the amino acid residue corresponding to phenylalanine 295 in Photinus pyralis wild-type luciferase or to leucine 297 in Luciola mingrelica, Luciola cruciata or Luciola lateralis wild-type luciferases, is mutated compared to the corresponding amino acid which appears in the corresponding wild-type luciferase sequence. The recombinant luciferase has increased thermostability compared to the corresponding wild-type luciferase.

1 . An isolated recombinant luciferase having luciferase activity and comprising a variant of wild-type  Photinus pyralis  luciferase of SEQ ID NO: 37, wherein the amino acid sequence of said recombinant luciferase has no more than 50 amino acid differences as compared to the amino acid sequence of SEQ ID NO: 37, wherein the recombinant luciferase has an amino acid substitution at the residue corresponding to position 35 of SEQ ID NO:37, and wherein the recombinant luciferase has increased thermostability as compared to the wild-type  Photinus pyralis  luciferase of SEQ ID NO:37. 
     
     
         2 . The recombinant luciferase of  claim 1 , wherein the amino acid residue corresponding to position 35 of SEQ ID NO:37 is alanine, valine, phenylalanine, isoleucine, proline, methionine or tryptophan. 
     
     
         3 . The recombinant luciferase of  claim 1 , wherein the amino acid residue corresponding to position 35 of SEQ ID NO:37 is alanine. 
     
     
         4 . An isolated nucleic acid which encodes the recombinant luciferase of  claim 1 . 
     
     
         5 . A vector comprising the nucleic acid of  claim 4 . 
     
     
         6 . An isolated cell transformed with the vector of  claim 5 . 
     
     
         7 . A bioluminescent assay comprising the steps of contacting the recombinant luciferase of  claim 1  with luciferin and detecting bioluminescence. 
     
     
         8 . A kit comprising the recombinant luciferase of  claim 1 .