US 2006/0068451 A1 · 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.

Claims24

Dependent claims

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.

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.

5 more elements

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.

5 more elements

Dependencies are inferred from the claim text.

Dimeric fluorescent polypeptides

Life of this patent

Dimeric fluorescent polypeptides

Life of this patent

Abstract

Claims24