Modified Fluorescent Proteins and Methods for Using Same

Abstract

Nucleic acid molecules encoding improved fluorescent mutants of the mKate2 protein, variants and derivatives thereof are provided, as well as proteins and peptides encoded by these nucleic acids. Also provided are proteins that are substantially similar to, or derivatives, homologues, or mutants of, the above-referenced specific proteins. Also provided are fragments of the nucleic acids and the peptides encoded thereby, as well as antibodies specific to the proteins and peptides of the invention. In addition, host-cells, stable cell lines and transgenic organisms comprising above-referenced nucleic acid molecules are provided. The subject protein and nucleic acid compositions find use in a variety of different applications and methods, particularly for labeling of biomolecules, cells or cell organelles. Finally, kits for use in such methods and applications are provided.

1 - 26 . (canceled) 
     
     
         27 . A nucleic acid present that encodes a fluorescent protein having a sequence identity of 90% or more to SEQ ID NO:30. 
     
     
         28 . The nucleic acid according to  claim 27 , wherein the protein includes one or more substitutions selected from the group consisting of R158A, R158S, R158D, R158N, R158G, K176E, K176N, K176D, K176S; Y195N, Y195D, Y195V, Y195A, Y195T, Y195E, Y195S, Y195C, Y195G; L225G, L225E, L225A, L225T, and L225N corresponding to SEQ ID NO:2. 
     
     
         29 . The nucleic acid according to  claim 28 , wherein the substitution is selected from the group consisting of R158A, K176E, Y195N, and L225G corresponding to SEQ ID NO:2. 
     
     
         30 . The nucleic acid according to  claim 29 , wherein the substitution is the substitution K176E corresponding to SEQ ID NO:2. 
     
     
         31 . The nucleic acid according to  claim 30 , further comprising substitutions R158A and Y195N corresponding to SEQ ID NO:2. 
     
     
         32 . The nucleic acid according to  claim 27 , further comprising a substitution of C-terminal residues corresponding to residues 223-232 of SEQ ID NO:2. 
     
     
         33 . The nucleic acid according to  claim 32 , wherein the C-terminal residues are substituted with SEQ ID NO:31. 
     
     
         34 . The nucleic acid according to  claim 27 , wherein the protein has the amino acid sequence of SEQ ID NO: 30. 
     
     
         35 . A vector comprising the nucleic acid of  claim 27 . 
     
     
         36 . The vector according to  claim 35 , wherein the vector is a plasmid. 
     
     
         37 . An expression cassette comprising:
 (a) the nucleic acid according to  claim 27 ; and   (a) a transcriptional initiation region that is operatively linked to the nucleic acid in (a) and is functional in an expression host; and (c) a transcriptional termination region that is operatively connected to the nucleic acid in (a) and is functional in the expression host.   
     
     
         38 . A host cell or progeny thereof, comprising the expression cassette according to  claim 37  as part of an extrachromosomal element or integrated into the genome of a host cell as a result of introduction of the expression cassette into the host cell. 
     
     
         39 . A transgenic cell, or progeny thereof, comprising the nucleic acid according to  claim 27 . 
     
     
         40 . A fluorescent protein having a sequence identity of 90% or more to SEQ ID NO:30. 
     
     
         41 . A fluorescent protein according to  claim 40 , wherein the fluorescent protein comprises a substitution is selected from the group consisting of R158A, R158S, R158D, R158N, R158G, K176E, K176N, K176D, K176S; Y195N, Y195D, Y195V, Y195A, Y195T, Y195E, Y195S, Y195C, Y195G; L225G, L225E, L225A, L225T, and L225N corresponding to SEQ ID NO:2. 
     
     
         42 . The fluorescent protein according to  claim 41 , wherein the substitution is selected from the group consisting of R158A, K176E, Y195N, and L225G corresponding to SEQ ID NO:2. 
     
     
         43 . The protein according to  claim 42 , wherein the substitution is the substitution K176E corresponding to SEQ ID NO:2. 
     
     
         44 . The protein according to  claim 41 , further comprising substitutions R158A and Y195N corresponding to SEQ ID NO:2. 
     
     
         45 . The protein according to  claim 40 , further comprising a substitution of C-terminal residues corresponding to residues 223-232 of SEQ ID NO:2. 
     
     
         46 . The protein according to  claim 45 , wherein the C-terminal residues are substituted with SEQ ID NO:31. 
     
     
         47 . A fluorescent protein having the sequence of SEQ ID NO:30. 
     
     
         48 . A kit comprising the nucleic acid according to  claim 27 . 
     
     
         49 . An antibody that specifically bind to the fluorescent protein of  claim 40 .