Dimeric fluorescent polypeptides

Abstract

The invention relates to proteins or polypeptides that comprise intramolecular dimers of fluorescent protein monomers. More specifically, the invention relates to recombinant polypeptides comprising a monomer of a fluorescent polypeptide, a linker peptide, and a second monomer of that fluorescent polypeptide, where the monomers form an intramolecular dimer. The invention also relates to nucleic acids encoding Intramolecular Dimer Fluorescent Proteins (IDFPs) and vectors comprising such nucleic acids. The invention further relates to methods of making IDFPs and methods of using them. IDFPs are, useful in any application suited for fluorescent proteins and are particularly useful in applications in which more than one fluorescent protein sharing complementary dimerization interfaces is present in the same mixture or is expressed in the same cell, because IDFPs do not form heterodimers.

1 . A polynucleotide encoding a recombinant fusion polypeptide comprising a first polypeptide peptide bonded to a second polypeptide, wherein said first and second polypeptides are found in nature as monomers of a multimeric protein, and wherein said recombinant fusion polypeptide is fluorescent when excited.  
     
     
         2 . The polynucleotide of  claim 1  wherein said first polypeptide and said second polypeptide are peptide bonded to each other via a linker sequence.  
     
     
         3 . The polynucleotide of  claim 2  wherein said linker sequence is from 5 to 50 amino acids long.  
     
     
         4 . The polynucleotide of  claim 1 , wherein said polynucleotide further encodes a third polypeptide peptide bonded to said recombinant fusion polypeptide.  
     
     
         5 . The polynucleotide of  claim 4  wherein said third polypeptide is a member of a specific binding pair.  
     
     
         6 . The polynucleotide of  claim 4  wherein said third polypeptide is fused to the amino terminus of said first polypeptide.  
     
     
         7 . The polynucleotide of  claim 4  wherein said third polypeptide is fused to the carboxy terminus of said second polypeptide.  
     
     
         8 . The polynucleotide of  claim 1  wherein each of said first and said second polypeptide, independently, is a monomer of a multimeric protein selected from the group consisting of  R. reniformis  GFP,  R. mulleri  GFP, and  A. victoria  GFP.  
     
     
         9 . The polynucleotide of  claim 1  wherein both of said first and second polypeptides are monomers of a single multimeric protein selected from the group consisting of  R. reniformis  GFP,  R. mulleri  GFP,  A. victoria  GFP.  
     
     
         10 . A vector comprising the polynucleotide of  claim 1 .  
     
     
         11 . A cell comprising the vector of  claim 10 .  
     
     
         12 . The cell of  claim 11 , said cell being a bacterial cell.  
     
     
         13 . The cell of  claim 11 , said cell being a eukaryotic cell.  
     
     
         14 . The eukaryotic cell of  claim 13 , wherein said cell is a yeast cell, an insect cell, or a mammalian cell.  
     
     
         15 . A method of producing a fluorescently labeled recombinant fusion polypeptide, said method comprising the steps of introducing a polynucleotide of  claim 1  to a cell, and culturing said cell under conditions that permit the synthesis of said recombinant fusion polypeptide, whereby said recombinant fusion polypeptide is produced.  
     
     
         16 . A method of labeling a cell with a fluorescent recombinant fusion polypeptide, said method comprising the steps of: 
 (a) introducing a polynucleotide of  claim 1  to a cell; and    (b) culturing said cell under conditions that permit the synthesis of said recombinant fusion polypeptide, whereby said cell is labeled with said fluorescent recombinant fusion polypeptide.    
     
     
         17 . The method of  claim 16  wherein, in said introducing step (a), said polynucleotide introduced to said cell further comprises a sequence encoding a third polypeptide fused in frame to the sequence encoding said recombinant fusion polypeptide.  
     
     
         18 . A method of monitoring the interaction of two polypeptides of interest, said method comprising the steps of: 
 (a) contacting a first polypeptide and a second polypeptide wherein: 
 (i) said first polypeptide is a recombinant fusion polypeptide comprising a first polypeptide bonded to a second polypeptide, wherein said first and second polypeptides are found in nature as monomers of a multimeric protein and said first and second polypeptides are not fluorescent donor and acceptor to each other, and wherein said recombinant fusion polypeptide is fluorescent when excited and further comprising a third polypeptide bonded to said recombinant fusion polypeptide wherein said third polypeptide is a first polypeptide of interest;  
 (ii) said second polypeptide comprises a second polypeptide of interest and is fluorescently labeled; and  
 (iii) the fluorophores comprised by said first and second polypeptides are fluorescent donor and fluorescent acceptor to each other;  
   (b) exciting said donor fluorophore; and    (c) detecting fluorescent emission from said fluorescent acceptor, wherein said emission is indicative of the interaction of said first and said second polypeptides of interest.    
     
     
         19 . The method of  claim 18  wherein said second polypeptide comprises a recombinant fusion polypeptide comprising a first polypeptide bonded to a second polypeptide, wherein said first and second polypeptides are found in nature as monomers of a multimeric protein and said first and second polypeptides are not fluorescent donor and acceptor to each other, and wherein said recombinant fusion polypeptide is fluorescent when excited and further comprising a third polypeptide bonded to said recombinant fusion polypeptide, wherein said third polypeptide is a member of a specific binding pair, wherein said third polypeptide of said second fusion polypeptide is different from said third polypeptide of said first fusion polypeptide.  
     
     
         20 . The method of  claim 18  wherein said contacting step is performed in vitro.  
     
     
         21 . The method of  claim 18  wherein said contacting step is performed in a cell.  
     
     
         22 . The method of  claim 21  wherein said contacting comprises the step of introducing nucleic acid encoding said first and said second polypeptides to a cell.  
     
     
         23 . A method of screening for a compound that modulates the interaction of a first and a second member of a specific binding pair, said method comprising the steps of: 
 (a) contacting a first polypeptide and a second polypeptide in the presence and absence of a candidate modulator wherein: 
 (i) said first polypeptide is a recombinant fusion polypeptide comprising a first polypeptide bonded to a second polypeptide, wherein said first and second polypeptides are found in nature as monomers of a multimeric protein and said first and second polypeptides are not fluorescent donor and acceptor to each other, and wherein said recombinant fusion polypeptide is fluorescent when excited and further comprising a third polypeptide bonded to said recombinant fusion polypeptide, wherein said third polypeptide is a member of a specific binding pair, wherein said member of a specific binding pair is said first member of a specific binding pair;  
 (ii) said second polypeptide is fluorescently labeled and comprises said second member of a specific binding pair; and  
 (iii) the fluorophores comprised by said first and second polypeptides are fluorescent donor and, acceptor to each other;  
   (b) exciting said donor fluorophore; and    (c) detecting the fluorescence of said acceptor fluorophore, wherein emission of the spectrum characteristic of said fluorescent acceptor indicates the interaction of said first and said second members of said specific binding pair, and wherein a change in said interaction in the presence of said candidate modulator indicates that said candidate modulator modulates the interaction of the members of said specific binding pair.    
     
     
         24 . The method of  claim 23  wherein said second polypeptide is a recombinant fusion polypeptide comprising a first polypeptide bonded to a second polypeptide, wherein said first and second polypeptides are found in nature as monomers of a multimeric protein and said first and second polypeptides are not fluorescent donor and acceptor to each other, and wherein said recombinant fusion polypeptide is fluorescent when excited and further comprising a third polypeptide bonded to said recombinant fusion polypeptide, wherein said third polypeptide is a member of a specific binding pair, and said member of a specific binding pair comprised by said second polypeptide is said second member of a specific binding pair.