Modified bioluminescent proteins and their use

Abstract

A modified bioluminescent protein responds to different physical, chemical, biochemical or biological conditions to produce light or radiation of altered characteristics when the bioluminescent reaction is initiated. The modified bioluminescent protein may respond to modification thereof, e.g. by covalent modification. The protein may inlcude signal peptides to “target” it. DNA coding for the bioluminescent protein may be altered to include tissue specific promoter or enhancer genes so that the altered DNA acts as reported gene.

What is claimed:  
     
         1 . A bioluminescent protein comprising a bioluminescent protein portion as a donor linked to an energy transfer protein portion as an acceptor by a link portion, said link portion comprising an interaction site for a selected analyte, wherein said analyte reacts with said interaction site, said bioluminescent protein being reactive in a bioluminescent reaction to produce light, such that the light is produced having a physical property of a first characteristic when the analyte is interacting with said interaction site, and is produced having a second different characteristic of said property when the analyte is not reacting with said interaction site.  
     
     
         2 . A bioluminescent protein according to  claim 1 , wherein said energy transfer protein portion and said bioluminescent protein portion are covalently bound via said linker portion.  
     
     
         3 . A bioluminescent protein according to  claim 1 , wherein said energy transfer protein portion and said bioluminescent protein portion are non-covalently bound via said linker portion.  
     
     
         4 . A bioluminescent protein according to  claim 1 , wherein said energy transfer protein portion comprises a green fluorescent protein, or a fluorescent domain thereof.  
     
     
         5 . A bioluminescent protein according to  claim 1 , wherein said energy transfer protein portion comprises a protein or a fluorescent domain thereof, which fluoresces at blue, yellow or higher waves lengths.  
     
     
         6 . A bioluminescent protein according to  claim 1 , wherein said linker portion comprises a nucleic acid molecule.  
     
     
         7 . A bioluminescent protein according to  claim 1 , wherein said linker portion comprises a protein or a fragment thereof.  
     
     
         8 . A bioluminescent protein according to  claim 1 , wherein said linker portion comprises a peptide.  
     
     
         9 . Nucleic acid coding for the bioluminescent protein as claimed in  claim 1 .  
     
     
         10 . A method of monitoring the interaction between a substrate and a substance to which said substrate is exposed, which comprises: 
 coupling a bioluminescent protein portion, capable of taking part in a bioluminescent reaction, to a first portion of said substrate,    coupling an energy transfer protein portion to a second portion of the substrate, whereby said bioluminescent protein portion and said energy transfer protein portion are in an energy transfer relationship,    exposing said substrate to said substance,    initiating said bioluminescent reaction, and    monitoring at least one of the amount of light emitted directly by said bioluminescent protein portion and the amount of light emitted by said energy transfer protein portion from energy transfer between said bioluminescent protein and said energy transfer protein portion.    
     
     
         11 . A method according to  claim 10 , wherein said substrate comprises a nucleic acid fragment containing one or more mutations or deletions and said substance comprises a nuclease responsive to said one or more mutations or deletions to cleave said nucleic acid fragment.  
     
     
         12 . A method of monitoring or detecting a substance in a sample, which comprises: 
 exposing said sample to: 
 a first reagent comprising a bioluminescent protein portion capable of taking part in a bioluminescent reaction and coupled to a first binding portion capable of binding specifically to a first region of said substance; and  
 a second reagent comprising an energy transfer protein portion coupled to a second binding portion capable of binding specifically to a second region of said substance;  
 allowing said binding to occur;  
 initiating said bioluminescent reaction, and,  
 monitoring at least one of the amount of liqht emitted directly by said bioluminescent protein portion and the amount of light emitted by said energy transfer protein portion on energy transfer between said bioluminescent protein portion and said energy transfer protein portion.  
   
     
     
         13 . A kit for use in monitoring or detecting a substance, which comprises: 
 a first reagent including a bioluminescent protein portion capable of taking part in a bioluminescent reaction coupled to a first specific binding portion capable of binding to a first region of said substance, and    a second reagent including an energy transfer protein portion coupled to a second specific binding portion capable of binding to a second region of said substance.    
     
     
         14 . A kit for use in monitoring or detecting a substance, which comprises: 
 nucleic acid coding for a first reagent comprising a bioluminescent protein portion coupled to a first specific binding portion capable of binding to a first region of said substance, and    nucleic acid coding for a second reagent comprising an energy transfer protein coupled to a second specific binding portion capable of binding to a second region of said substance.    
     
     
         15 . A method of monitoring a sample for the presence of a nucleic acid fragment of interest, which comprises: 
 introducing into said sample a first nucleic acid amplification primer having coupled thereto nucleic acid coding for a bioluminescent protein, and a second nucleic acid primer having coupled thereto nucleic acid coding for an energy transfer protein or a fluorescent domain thereof;    initiating said nucleic acid amplification process;    expressing the product of said nucleic acid amplification process to obtain one or more bioluminescent proteins;    initiating one or more bioluminescent reactions involving said one or more bioluminescent proteins, and monitoring the characteristics of the light emitted during said bioluminescent reaction.    
     
     
         16 . A method of monitoring or detecting a substance in a sample, which comprises; 
 exposing said sample to: 
 a bioluminescent protein comprising a bioluminescent protein portion as a donor linked to an energy transfer protein portion as an acceptor by a link portion, said link portion comprising an interaction site for said substance, wherein said substance reacts with said interaction site, said bioluminescent protein being reactive in a bioluminescent reaction to produce light, such that the light is produced having a physical property of a first characteristic when the substance is interacting with said interaction site, and is produced having a second different characteristic of said property when the analyte is not reacting with said interaction site,  
 initiating said bioluminescent reaction, and  
 monitoring at least in of the light emitted directly by said bioluminescent protein portion and the light emitted by said energy transfer protein portion on energy transfer between said bioluminescent protein portion and said energy transfer protein portion.  
   
     
     
         17 . A kit for use in detecting a substance of interest, which comprises: 
 a first reagent comprising a first fragment of nucleic acid coding for a bioluminescent protein portion, said first nucleic acid fragment being coupled to a first nucleic acid amplification primer for hybridizing to said nucleic acid sample, and    a second reagent comprising a second nucleic acid fragment coding for an energy transfer protein, said second nucleic acid fragment being coupled to a second nucleic acid amplification primer capable of hybridizing to a second region of said nucleic acid target.    
     
     
         18 . A method of monitoring the interaction between a substrate and a substance to which said substrate is exposed, which comprises: 
 coupling a bioluminescent protein portion, capable of taking part in a bioluminescent reaction, to a first portion of said substrate,    coupling an energy transfer protein portion to a second portion of the substrate, whereby said bioluminescent protein portion and said energy transfer protein portion are in an energy transfer relationship,    exposing said substrate to said substance,    initiating said bioluminescent reaction, and    monitoring at least one of the amount of light emitted directly by said bioluminescent protein portion and the amount of light emitted by said energy transfer protein portion resulting from energy transfer between said bioluminescent protein portion and said energy transfer protein portion, wherein said substrate comprises a nucleic acid fragment containing one or more mutations or deletions and said substance comprises a nuclease responsive to said one or more mutations or deletions to cleave said nucleic acid fragment.