Monomeric and dimeric fluorescent protein variants and methods for making same

Abstract

The present invention relates generally to variant fluorescent proteins, and more specifically to monomeric and dimeric forms of Anthozoan fluorescent proteins. In one aspect, the present invention provides variants of fluorescent proteins, where the variants have a reduced propensity to tetramerize, and form dimeric or monomerc structures. The invention also relates to methods of making and using such fluorescent protein monomers and dimers.

1 . A polynucleotide sequence encoding a Discosoma red fluorescent protein (DsRed) variant having a reduced propensity to oligomerize, comprising one or more amino acid substitutions at the AB interface, at the AC interface, or at the AB and AC interfaces of the wild-type DsRed amino acid sequence of SEQ ID NO: 1, where the substitutions result in reduced propensity of the DsRed variant to form tetramers, wherein said variant displays detectable fluorescence of at least one red wavelength.  
     
     
         2 . The polynucleotide sequence of  claim 1 , wherein said variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 1.  
     
     
         3 . The polynucleotide sequence of  claim 1 , wherein said detectable fluorescence matures at a rate at least about 80% as fast as the rate of fluorescence maturation of wild-type DsRed of SEQ ID NO: 1.  
     
     
         4 . The polynucleotide sequence of  claim 1  having improved fluorescence maturation relative to DsRed of SEQ ID NO: 1.  
     
     
         5 . The polynucleotide sequence of  claim 1  substantially retaining the fluorescing properties of DsRed of SEQ ID NO: 1.  
     
     
         6 . The polynucleotide sequence of  claim 1 , which has a propensity to form a dimer.  
     
     
         7 . The polynucleotide sequence of  claim 6 , which has substitutions in the AB interface and forms an AC dimer.  
     
     
         8 . The polynucleotide sequence of  claim 6  comprising at least nine amino acid substitutions that are at residues 2, 5, 6, 21, 41, 42, 44, 117, and 217, and additionally at least one more substitution including substitution at residue 125 of SEQ ID NO: 1.  
     
     
         9 . The polynucleotide sequence of  claim 8 , optionally further comprising at least one additional amino acid substitution that is at residue 71, 118, 163, 179, 197, 127, or 131 of SEQ ID NO: 1.  
     
     
         10 . The polynucleotide sequence of  claim 9 , wherein any one or more of said substitutions is optionally selected from R2A, K5E, N6D, T21S, H41T, N42Q, V44A, V71A, C117T, F118L, I125R, V127T, S131P, K163Q/M, S179T, S197T, and T217A/S.  
     
     
         11 . The polynucleotide sequence of  claim 1 , wherein said variant is selected from the group of variants consisting of dimer1, dimer1.02, dimer1.25, dimer1.26, dimer 1.28, dimer1.34, dimer1.56, dimer1.61, dimer1.76, provided in  FIG. 20 .  
     
     
         12 . The polynucleotide sequence of  claim 1  that is dimer2 (SEQ ID NO: 6).  
     
     
         13 . The polynucleotide sequence of  claim 6 , having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 1.  
     
     
         14 . The polynucleotide sequence of  claim 6 , having at least about 95% sequence identity with the amino acid sequence of SEQ ID NO: 1.  
     
     
         15 . The polynucleotide sequence of  claim 1 , which is a monomer.  
     
     
         16 . The polynucleotide sequence of  claim 15 , which has substitutions in the AB interface and the AC interface.  
     
     
         17 . The polynucleotide sequence of  claim 15  comprising at least 14 amino acid substitutions that are at residues 2, 5, 6, 21, 41, 42, 44, 71, 117, 127, 163, 179, 197, and 217, and additionally at least one more substitution that is at residue 125 of SEQ ID NO: 1.  
     
     
         18 . The polynucleotide sequence of  claim 17 , optionally further comprising at least one additional amino acid substitution at residue 83, 124, 125, 150, 153, 156, 162, 164, 174, 175, 177, 180, 192, 194, 195, 222, 223, 224, and 225 of SEQ ID NO: 1.  
     
     
         19 . The polynucleotide sequence of  claim 18 , wherein any one or more of said substitutions is optionally selected from R2A, K5E, N6D, T21S, H41T, N42Q, V44A, V71A, K83L, C117E/T, F124L, I125R, V127T, L150M, R153E, V156A, H162K, K163Q/M, L174D, V175A, F177V, S179T, I180T, Y192A, Y194K, V195T, S197A/T/I, T217A/S, H222S, L223T, F224G, L225A  
     
     
         20 . The polynucleotide sequence of  claim 15 , wherein said variant is selected from the group of variants consisting of mRFP0.1, mRFP0.2, mRFP0.3, mRFP0.4a, mRFP0.4b, mP11, mP17, m1.01, m1.02, mRFP0.5a, m1.12, mRFP0.5b, m1.15, m1.19, mRFP0.6, m124, m131, m141, m163, m173, m187, m193, m200, m205 and m220, provided in  FIG. 20 .  
     
     
         21 . The polynucleotide sequence of  claim 15  that is mRFP1 (SEQ ID NO: 8).  
     
     
         22 . The polynucleotide sequence of  claim 15 , having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 1.  
     
     
         23 . The polynucleotide sequence of  claim 15 , having at least about 95% sequence identity with the amino acid sequence of SEQ ID NO: 1.  
     
     
         24 . A polynucleotide sequence encoding a tandem dimer comprising two DsRed protein variants encoded by the polynucleotide sequence of  claim 1 , operatively linked by a peptide linker.  
     
     
         25 . The polynucleotide sequence of  claim 24 , wherein said peptide linker is about 10 to about 25 amino acids long.  
     
     
         26 . The polynucleotide sequence of  claim 25 , wherein said peptide linker is about 12 to about 22 amino acids long.  
     
     
         27 . The polynucleotide sequence of  claim 24 , wherein said peptide linker is selected from the group consisting of GHGTGSTGSGSS (SEQ ID NO: 17), RMGSTSGSTKGQL (SEQ ID NO: 18), and RMGSTSGSGKPGSGEGSTKGQL (SEQ ID NO: 19).  
     
     
         28 . The polynucleotide sequence of  claim 24  wherein at least one of said DsRed subunits is selected from the group consisting of dimer1, dimer1.02, dimer1.25, dimer1.26, dimer1.28, dimer1.34, dimer1.56, dimer1.61, dimer1.76, and dimer2, provided in  FIG. 20 .  
     
     
         29 . The polynucleotide sequence of  claim 24  wherein said tandem dimer is a homodimer.  
     
     
         30 . The polynucleotide sequence of  claim 24  wherein said tandem dimer is a heterodimer.  
     
     
         31 . The polynucleotide sequence of  claim 24  wherein at least one of said DsRed variants is dimer2 (SEQ ID NO: 6).  
     
     
         32 . A polynucleotide sequence encoding a fusion protein, comprising at least one DsRed protein variant encoded by the polynucleotide sequence of  claim 1  operatively joined to at least one polypeptide of interest.  
     
     
         33 . The polynucleotide sequence of  claim 32 , wherein said fusion protein comprises a peptide tag.  
     
     
         34 . The polynucleotide sequence of  claim 33 , wherein the peptide tag is a polyhistidine peptide tag.  
     
     
         35 . A polynucleotide sequence comprising a DsRed protein monomeric variant encoded by the polynucleotide sequence of  claim 15  operatively joined to at least one polypeptide of interest.  
     
     
         36 . The polynucleotide sequence of  claim 35 , wherein said fusion protein comprises a peptide tag.  
     
     
         37 . The polynucleotide sequence of  claim 36 , wherein the peptide tag is a polyhistidine peptide tag.  
     
     
         38 . The DsRed protein variant encoded by the polynucleotide sequence of  claim 1 .  
     
     
         39 . The DsRed protein variant encoded by the polynucleotide sequence of  claim 6 .  
     
     
         40 . The DsRed protein variant encoded by the polynucleotide sequence of  claim 15 .  
     
     
         41 . The DsRed protein variant encoded by the polynucleotide sequence of  claim 24 .  
     
     
         42 . A kit, comprising at least one polynucleotide sequence of  claim 1 .  
     
     
         43 . A kit, comprising at least one polypeptide encoded by the polynucleotide sequence of  claim 1 .  
     
     
         44 . A vector comprising the polynucleotide sequence of  claim 1 .  
     
     
         45 . A vector comprising the polynucleotide sequence of  claim 6 .  
     
     
         46 . A vector comprising the polynucleotide sequence of  claim 15 .  
     
     
         47 . A vector comprising the polynucleotide sequence of  claim 41 .  
     
     
         48 . The vector of  claim 46 , wherein the vector is an expression vector.  
     
     
         49 . A host cell comprising the vector of  claim 44 .  
     
     
         50 . A host cell comprising the vector of  claim 45 .  
     
     
         51 . A host cell comprising the vector of  claim 46 .  
     
     
         52 . A host cell comprising the vector of  claim 47 .  
     
     
         53 . A host cell comprising the expression vector of  claim 48 .  
     
     
         54 . A method for the generation of a dimeric or monomeric variant of a fluorescent protein which has propensity to tetramerize or dimerize, comprising: 
 (a) mutagenizing at least one amino acid residue in said fluorescent protein to produce a dimeric variant, if the protein had the propensity to tetramerize, and a monomeric variant, if the protein had the propensity to dimerize; and    (b) mutagenizing at least one additional amino acid residue to yield a dimeric or monomeric variant, which retains the qualitative ability to fluoresce in the same wavelength region as the non-mutagenized fluorescent protein.    
     
     
         55 . The method of  claim 54 , further comprising the step of introducing a further mutation into a dimeric variant produced from a fluorescent protein that had the propensity to form tetramers to produce a monomeric variant.  
     
     
         56 . The method of  claim 55 , wherein said further step follows (a).  
     
     
         57 . The method of  claim 54 , wherein said dimeric or monomeric variant has improved fluorescence intensity or fluorescence maturation relative to the non-mutagenized fluorescent protein.  
     
     
         58 . The method of  claim 54 , wherein said mutagenizing is by multiple overlap extension with semidegenerate primers.  
     
     
         59 . The method of  claim 54 , wherein said mutagenizing is by error-prone PCR.  
     
     
         60 . The method of  claim 54 , wherein said mutagenizing is by site directed mutagenesis.  
     
     
         61 . The method of  claim 54 , wherein said mutagenizing is by a combination of at least two of multiple overlap extension, error-prone PCR and site directed mutagenesis.  
     
     
         62 . The method of  claim 54 , wherein said fluorescent protein is an Anthozoan fluorescent protein.  
     
     
         63 . The method of  claim 62  wherein said Anthozoan fluorescent protein fluoresces at a red wavelength.  
     
     
         64 . The method of  claim 62  wherein said Anthozoan fluorescent protein is  Discosoma  DsRed.  
     
     
         65 . The method of  claim 54 , wherein said variant fluorescent protein has a propensity to form dimers.  
     
     
         66 . The method of  claim 54 , wherein said variant fluorescent protein has a propensity to form monomers.  
     
     
         67 . A method for the detection transcriptional activity, comprising 
 (a) providing a host cell comprising a vector, wherein said vector comprises nucleotide sequence encoding a variant DsRed fluorescent protein of  claim 1  operably linked to at least one expression control sequence, and a means to assay said variant fluorescent protein fluorescence, and    (a) assaying fluorescence of said variant fluorescent protein produced by said host cell, where variant fluorescent protein fluorescence is indicative of transcriptional activity.    
     
     
         68 . A polynucleotide sequence encoding a polypeptide probe suitable for use in fluorescence resonance energy transfer (FRET), comprising at least one polynucleotide of  claim 1 .  
     
     
         69 . A method for the analysis of in vivo localization or trafficking of a polypeptide of interest, comprising the steps of: 
 (a) providing a polynucleotide sequence of  claim 32  and a host cell or tissue, and    (b) visualizing said fusion protein that is expressed in said host cell or tissue.