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 Phtotinus pyralis, Luciola mingrelica, Luciola cruciata, Luciola lateralis, Hotaria parvula, Pyrophorus plagiophthalamus, Lampyris noctiluca, Pyrocoelia miyako or Photinus pennsylvanica . In the seguence 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 Photlnus 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  295  of SEQ ID NO: 37, and wherein the recombinant luciferase has increased thermostability as compared to the wild-type Photlnus pyralis luciferase of SEQ ID NO: 37. 
     
     
         2 . The recombinant luciferase of  claim 1 , further comprising an amino acid other than phenylalanine at the amino acid residue corresponding to position  14  of SEQ ID NO: 37. 
     
     
         3 . The recombinant luciferase of  claim 2 , wherein the amino acid residue corresponding to position  14  of SEQ ID NO:  37  is alanine. 
     
     
         4 . The recombinant luciferase of  claim 1 , further comprising an amino acid other than leucine at the amino acid residue corresponding to position  35  of SEQ ID NO: 37. 
     
     
         5 . The recombinant luciferase of  claim 4 , wherein the amino acid residue corresponding to position  35  of SEQ ID NO: 37 is alanine. 
     
     
         6 . The recombinant luciferase of  claim 1 , further comprising an amino acid other than glutamic acid at the amino acid residue corresponding to position  354  of SEQ ID NO: 37. 
     
     
         7 . The recombinant luciferase of  claim 6 , wherein the amino acid residue corresponding to position  354  of SEQ ID NO: 37 is lysine. 
     
     
         8 . The recombinant luciferase of  claim 1 , further comprising an amino acid other than, alanine at the amino acid residue corresponding to position  215  of SEQ ID NO: 37. 
     
     
         9 . The recombinant luciferase of  claim 8 , wherein the amino acid residue corresponding to position  215  of SEQ ID NO: 37 is lysine. 
     
     
         10 . The recombinant luciferase of  claim 1 , further comprising an amino acid other than threonine at the amino acid residue corresponding to position  214  of SEQ ID NO: 37, an amino acid other than isoleucine at the amino acid residue corresponding to position  232  of SEQ ID NO: 37, and an amino acid other than glutamic acid at the amino acid residue corresponding to position  354  of SEQ ID NO: 37. 
     
     
         11 . The recombinant luciferase of  claim 10 , wherein the amino acid corresponding to position  214  of SEQ ID NO: 37 is alanine, the amino acid corresponding to position  232  of SEQ ID NO: 37 is alanine and the amino acid corresponding to position  354  of SEQ ID NO: 37 is lysine. 
     
     
         12 . The recombinant luciferase of  claim 11 , further comprising an amino acid other than phenylalanine at the amino acid residue corresponding to position.  14  of SEQ ID NO: 37 and an amino acid other than, leucine at the amino acid residue corresponding to position  35  of SEQ ID NO: 37. 
     
     
         13 . The recombinant luciferase of  claim 12 , wherein the amino acid, corresponding to position  14  of SEQ ID NO: 37 is alanine and the amino acid corresponding to position  35  of SEQ ID NO: 37 is alanine, 
     
     
         14 . The recombinant luciferase of  claim 12 , further comprising an amino acid other than alanine at the amino acid residue corresponding to position  215  of SEQ ID NO: 37. 
     
     
         15 . The recombinant luciferase of  claim 14 , wherein the amino acid corresponding to position  215  of SEQ ID NO: 37 is leucine. 
     
     
         16 . An. isolated nucleic acid which encodes the recombinant luciferase of  claim 1 . 
     
     
         17 . A vector comprising the nucleic acid of  claim 16 . 
     
     
         18 . An Isolated cell transformed with the vector of  claim 17 . 
     
     
         19 . A bioluminescent assay comprising the steps of contacting the recombinant luciferase of  claim 1  with luciferin and detecting bioluminescence. 
     
     
         20 . A kit comprising the recombinant luciferase of  claim 1 .