Mutant luciferases

Abstract

The invention provides active, non-naturally occurring mutants of beetle luciferases and DNAs which encode such mutants. A mutant luciferase of the invention differs from the corresponding wild-type luciferase by producing bioluminescence with a wavelength of peak intensity that differs by at least 1 nm from the wavelength of peak intensity of the bioluminescence produced by the wild-type enzyme. The mutant luciferases and DNAs of the invention are employed in various biosensing applications.

We claim:  
     
         1 . An isolated DNA molecule comprising a segment having a sequence which encodes a synthetic mutant beetle luciferase comprising an amino acid sequence that differs from that of the corresponding wild-type luciferase by at least one amino acid substitution, the position of the amino acid substitution corresponding to a position in the amino acid sequence of LucPplGR of SEQ ID NO:2 selected from the group consisting of position 215, 224, 238, 242, 247 and 348, wherein the mutant luciferase produces bioluminescence having a shift in wavelength of peak intensity of at least 1 nanometer relative to the bioluminescence produced by the wild-type luciferase, wherein for the encoded amino acid sequence, the corresponding wild-type luciferase is LucPplGR of SEQ ID NO:2, and wherein the encoded synthetic mutant luciferase includes an amino acid substitution selected from the group consisting of LucPplGR-R 215 Q, -R 215 S, -R 215 Y, -R 215 K, -R 215 C, -R 215 E, -R 215 F, -H 242 S, -H 348 A, -V 224 F/L 238 M, and -V 224 F/S 247 Y.  
     
     
         2 . An isolated DNA molecule according to  claim 1 , wherein the encoded mutant luciferase has one amino acid substitution.  
     
     
         3 . An isolated DNA molecule according to  claim 1 , wherein the encoded mutant luciferase has two amino acid substitutions.  
     
     
         4 . An isolated DNA molecule according to  claim 1 , wherein the encoded synthetic mutant luceriferase is LucPplGR-R 215 Q.  
     
     
         5 . An isolated DNA molecule according to  claim 1 , wherein encoded synthetic mutant luceriferase is LucPplGR-R 215 S.  
     
     
         6 . An isolated DNA molecule according to  claim 1 , wherein the encoded synthetic mutant luceriferase is LucPplGR-R 215 Y.  
     
     
         7 . An isolated DNA molecule according to  claim 1 , wherein the encoded synthetic mutant luceriferase is LucPplGR-R 215 K.  
     
     
         8 . An isolated DNA molecule according to  claim 1 , wherein the encoded synthetic mutant luceriferase is LucPplGR-R 215 C.  
     
     
         9 . An isolated DNA molecule according to  claim 1 , wherein the encoded synthetic mutant luceriferase is LucPplGR-R 215 E.  
     
     
         10 . An isolated DNA molecule according to  claim 1 , wherein the encoded synthetic mutant luceriferase is LucPplGR-R 215 F.  
     
     
         11 . An isolated DNA molecule according to  claim 1 , wherein the encoded synthetic mutant luceriferase is LucPplGR-H 242 S.  
     
     
         12 . An isolated DNA molecule according to  claim 1 , wherein the encoded synthetic mutant luceriferase is LucPplGR-H 348 A.  
     
     
         13 . An isolated DNA molecule according to  claim 1 , wherein the encoded synthetic mutant luceriferase is LucPplGR-V 224 F/L 238 M.  
     
     
         14 . An isolated DNA molecule according to  claim 1 , wherein the encoded synthetic mutant luceriferase is LucPplGR-V 224 F/S 247 Y.  
     
     
         15 . An isolated DNA molecule comprising a segment having a sequence which encodes a mutant beetle luciferase having an amino acid sequence that differs from that of the corresponding wild-type luciferase LucPplGR by at least one amino acid substitution, wherein the encoded mutant luciferase is selected from the group consisting of LucPplGR-R 215 Q, -R 215 S, -R 215 Y, -R 215 K, -R 215 C, -R 215 E, -R 215 F, -H 242 S, -H 238 A, -V 224 F/L 238 M, and -V 224 F/S 247 Y and the encoded mutant luciferase produces bioluminescence having a shift in wavelength of peak intensity of at least 1 nanometer relative to the bioluminescence produced by the wild-type luciferase.