Modified Luciferases And Uses Thereof

Abstract

The present invention encompasses modified luciferases, methods for making modified luciferases, and assays utilizing modified luciferases. Modified luciferases of the invention show increased activity over wildtype luciferases and also show increased stability of signal. The present invention also encompasses multiplex assays utilizing multiple luciferases with different emission spectra.

What is claimed is: 
     
         1 . An isolated polynucleotide that encodes a modified  L. Italica  luciferase, wherein said modified  L. Italica  luciferase shows increased luciferase activity when expressed in mammalian cells as compared to a non human codon optimized mutant  L. Italica  luciferase. 
     
     
         2 . The isolated polynucleotide of  claim 1 , wherein said modified  L. Italica  luciferase shows approximately 1000-fold increased luciferase activity when expressed in mammalian cells as compared to a non human codon optimized mutant  L. Italica  luciferase. 
     
     
         3 . The isolated polynucleotide of  claim 1 , wherein said modified  L. Italica  luciferase is a red-emitting luciferase with an emission maximum of approximately 617 nm. 
     
     
         4 . The isolated polynucleotide of  claim 1 , wherein said modified  L. Italica  luciferase is human codon-optimized. 
     
     
         5 . The isolated polynucleotide of  claim 1 , wherein said modified  L. Italica  luciferase is a green-emitting luciferase with an emission maximum of approximately 550 nm. 
     
     
         6 . The isolated polynucleotide of  claim 1 , wherein said  L. Italica  luciferase comprises a secretory signal at its amino terminal end. 
     
     
         7 . The isolated polynucleotide of  claim 6 , wherein said secretory signal is a chymotrypsinogen secretory signal. 
     
     
         8 . An assay utilizing the modified  L. Italica  luciferase of  claim 1 . 
     
     
         9 . The assay of  claim 8 , wherein said assay is a multiplexed reporter assay. 
     
     
         10 . An isolated polynucleotide that encodes a modified  Renilla  luciferase, wherein said modified  Renilla  luciferase shows increased activity and stability over a native human codon optimized  Renilla  luciferase. 
     
     
         11 . The isolated polynucleotide of  claim 10 , wherein said modified  Renilla  luciferase is a green-emitting  Renilla  luciferase. 
     
     
         12 . The isolated polynucleotide of  claim 10 , wherein said modified  Renilla  luciferase comprises a secretory signal at its amino terminal end. 
     
     
         13 . A multiplexed luciferase assay comprising at least two different luciferase reports, wherein said at least two different luciferase reporters emit at two different wavelengths and/or utilize different substrates.