Luciferase mutant

Abstract

A recombinant protein having luciferase activity and at least 60% similarity to a wild-type luciferase wherein in the sequence of the enzyme, the amino acid residue corresponding to residue 357 in Photinus pyralis luciferase is mutated as compared to the corresponding wild-type luciferase, such that the luciferase enzyme is able to emit light at a different wavelength as compared to the corresponding wild-type luciferase and/or has enhanced thermostability as compared to the corresponding wild-type luciferase. In general, the residue corresponding to 357 in Photinus pyralis luciferase is changed from an acidic amino acid to a non-acidic amino acid, and preferably an uncharged polar amino acid such as tyrosine. Mutant luciferases in accordance with the invention can produce a large (50 nm) wavelength shift in emitted light and have good thermostability. The resultant colour shift can be reversed by addition of coenzyme A. These properties make the mutant particularly useful in a variety of assays.

1 . A recombinant protein having luciferase activity and comprising an amino acid sequence with at least 90% sequence similarity to a wild-type luciferase from  Photuris pennslyvanica , wherein, in the amino acid sequence of the recombinant protein, the amino acid residue corresponding to residue 357 of the amino acid sequence of  Photinus pyralis  luciferase is mutated compared to the wild-type luciferase, and the recombinant protein emits light at a different wavelength and/or has enhanced thermostability as compared to wild-type  Photuris pennsylvanica.    
     
     
         2 . The recombinant protein of  claim 1 , wherein the amino acid residue corresponding to residue 357 in  Photinus pyralis  luciferase is mutated to something other than valine. 
     
     
         3 . The recombinant protein of  claim 2 , wherein the amino acid residue corresponding to residue 357 in  Photinus pyralis  luciferase is mutated to tyrosine, phenylalanine, tryptophan, lysine, asparagine, or isoleucine. 
     
     
         4 . A nucleic acid which encodes the recombinant luciferase protein of  claim 1 . 
     
     
         5 . The nucleic acid of  claim 4  which comprises a synthetic nucleic acid. 
     
     
         6 . The nucleic acid of  claim 4  wherein the codon usage has been optimized for an  Escherichia coli  expression host and/or unique restriction sites have been introduced. 
     
     
         7 . A vector comprising the nucleic acid of  claim 4 . 
     
     
         8 . An isolated cell transformed with the vector of  claim 7 . 
     
     
         9 . The recombinant luciferase protein of  claim 1  wherein the recombinant protein has enhanced thermostability as compared to wild-type  Photuris pennsylvanica  luciferase. 
     
     
         10 . The recombinant luciferase protein of  claim 1  further comprising at least one mutation compared to the wild-type luciferase at
 a) the amino acid residue corresponding to residue 354 of the amino acid sequence of  Photinus pyralis  luciferase; 
 b) the amino acid residue corresponding to residue 215 of the amino acid sequence of  Photinus pyralis  luciferase; 
 c) the amino acid residue corresponding to residue 214 of the amino acid sequence of  Photinus pyralis  luciferase; 
 d) the amino acid residue corresponding to residue 232 of the amino acid sequence of  Photinus pyralis  luciferase; 
 e) the amino acid residue corresponding to residue 295 of the amino acid sequence of  Photinus pyralis  luciferase; 
 f) the amino acid residue corresponding to residue 14 of the amino acid sequence of  Photinus pyralis  luciferase; 
 g) the amino acid residue corresponding to residue 35 of the amino acid sequence of  Photinus pyralis  luciferase; 
 h) the amino acid residue corresponding to residue 105 of the amino acid sequence of  Photinus pyralis  luciferase; 
 i) the amino acid residue corresponding to residue 234 of the amino acid sequence of  Photinus pyralis  luciferase; 
 j) the amino acid residue corresponding to residue 420 of the amino acid sequence of  Photinus pyralis  luciferase; or 
 k) the amino acid residue corresponding to residue 310 of the amino acid sequence of  Photinus pyralis  luciferase; 
 
     
     
         11 . The recombinant luciferase protein of  claim 10  wherein the recombinant protein has enhanced thermostability as compared to wild-type  Photuris pennslyvanica  luciferase. 
     
     
         12 . A nucleic acid which encodes the recombinant luciferase protein of  claim 10 . 
     
     
         13 . The nucleic acid of  claim 12  which comprises a synthetic nucleic acid. 
     
     
         14 . The nucleic acid of  claim 12  wherein the codon usage has been optimized for an  Escherichia coli  expression host and/or unique restriction sites have been introduced. 
     
     
         15 . A vector comprising the nucleic acid of  claim 12 . 
     
     
         16 . An isolated cell transformed with the vector of  claim 15 .