Mutant blue fluorescent protein and method of using the same for fluorescence resonance energy transfer and blue fluorescent fish

Abstract

The present invention discloses a mutant blue fluorescent protein (BFP) exhibiting, at an aerobic or anaerobic system, larger fluorescent intensity than a BFPvv D7 of SEQ ID NO:2 derived from a wild type BFP, BfgV of SEQ ID NO:1, obtained from Vibrio vulnificus , wherein a set of mutation positions of the mutant BFP corresponding to SEQ ID NO:2 comprises position 176 or position 178. In a preferred embodiment, the set of mutation positions of the mutant BFP corresponding to SEQ ID NO:2 comprises a S176R mutation or a V178I mutation. Moreover, methods of using the blue fluorescent proteins from Vibrio vulnificus for fluorescence resonance energy transfer (FRET) and a blue fluorescent fish are also provided.

1 . A mutant blue fluorescent protein (BFP) exhibiting, at an aerobic or anaerobic system, larger fluorescent intensity than a BFPvv D7 of SEQ ID NO:2 derived from a wild type BFP, BfgV of SEQ ID NO:1, obtained from  Vibrio vulnificus , wherein a set of mutation positions of the mutant BFP corresponding to SEQ ID NO:2 comprises position 176 or position 178. 
     
     
         2 . The mutant BFP as claimed in  claim 1 , wherein the set of mutation positions of the mutant BFP corresponding to SEQ ID NO:2 comprises a S176R mutation or a V178I mutation. 
     
     
         3 . The mutant BFP as claimed in  claim 1 , comprising the amino acid sequence set forth in SEQ ID NO:3. 
     
     
         4 . The mutant BFP as claimed in  claim 1 , the fluorescent intensity of which is stable at 37° C. 
     
     
         5 . The mutant BFP as claimed in  claim 1 , having a propensity to form a monomer. 
     
     
         6 . The mutant BFP as claimed in  claim 1 , fluorescing through binding to NADPH with no need of undergoing a maturation step before emitting fluorescence. 
     
     
         7 . The mutant BFP as claimed in  claim 1 , having fluorescence spectra with an excitation peak at 352 nm and an emission peak at 440 nm. 
     
     
         8 . The mutant BFP as claimed in  claim 1 , which is 1.2-4 times the fluorescent intensity of the BFPvv D7 of SEQ ID NO:2. 
     
     
         9 . The mutant BFP as claimed in  claim 1 , mutated by an error-prone PCR method or a DNA shuffling method with using the BFPvv D7 of SEQ ID NO:2 as parents. 
     
     
         10 . The mutant BFP as claimed in  claim 1 , comprising to be for use as a cell marker or protein expression indicator in a prokaryotic or eukaryotic cell. 
     
     
         11 . A nucleic acid comprising a sequence encoding the mutant BFP as claimed in  claim 1 . 
     
     
         12 . A host cell stably transformed or transfected with the nucleic acid as claimed in  claim 11 . 
     
     
         13 . The host cell as claimed in  claim 12 , wherein the host cell expresses the mutant BFP encoded by the nucleic acid. 
     
     
         14 . The host cell as claimed in  claim 12 , wherein the host cell comprises a prokaryotic cell or a eukaryotic cell. 
     
     
         15 . A method of using a blue fluorescent protein (BFP) for fluorescence resonance energy transfer (FRET), comprising using the mutant BFP, the BFPvv D7 of SEQ ID NO:2, or the BfgV of SEQ ID NO:1 as claimed in  claim 1  as a fluorophore. 
     
     
         16 . The method of using the BFP for FRET as claimed in  claim 15 , wherein the mutant BFP, the BFPvv D7 of SEQ ID NO:2, or the BfgV of SEQ ID NO:1 as claimed in  claim 1  is used as a donor fluorophore and coupled with one of several green fluorescent protein (GFP) variants as an acceptor fluorophore. 
     
     
         17 . The method of using the BFP for FRET as claimed in  claim 16 , suitable for use in detecting a Ca 2+  concentration or caspase-3 activation in an apoptotic cell. 
     
     
         18 . A method of using a blue fluorescent protein (BFP) for producing a blue fluorescent fish, comprising using the mutant BFP, the BFPvv D7 of SEQ ID NO:2, or the BfgV of SEQ ID NO:1 as claimed in  claim 1  as a fluorophore by a transgenic technology.