Fluorescent proteins and related methods and compounds
The invention includes fusion polypeptides including a first fluorescent protein, e.g., a FRET donor protein, a second fluorescent protein, e.g., a FRET acceptor protein, and, linked to at least one of the fluorescent (e.g., FRET donor or FRET acceptor) proteins, an Fc-region of an immunoglobulin. The polypeptide can be immobilized with respect to a surface via the Fc-region even in the absence of antibodies to either the FRET donor protein or FRET acceptor protein, and can be used as a calibration standard for fluorescence resonance energy transfer includes a polypeptide.
1. A composition comprising:
a plurality of beads having different fluorescence resonance energy transfer (FRET) efficiencies, wherein immobilized on each of the beads are fusion proteins comprising:
first and second fluorescent proteins, wherein the first and second fluorescent proteins are a FRET pair;
a linker of a known length between the first and second fluorescent proteins; and
an Fc region linked to one of the fluorescent proteins,
the plurality of beads comprising fusion proteins with linkers of varied lengths to produce the different FRET efficiencies,
wherein each bead comprises a marker so that beads with different FRET efficiencies can be identified in a mixture.
2. The composition of claim 1 , wherein at least one of the first and second fluorescent proteins is a green fluorescent protein or variant thereof.
3. The composition of claim 2 , wherein the green fluorescent protein or variant thereof is selected from the group consisting of green fluorescent protein (GFP), cyan fluorescent protein (CFP), photoactivatable green fluorescent protein (paGFP), and yellow fluorescent protein (YFP).
4. The composition of claim 1 , wherein at least one of the first and second fluorescent proteins is a reef coral fluorescent protein (RCFP) or variant thereof.
5. The composition of claim 4 , wherein the fluorescent protein is selected from Anemonia majano cyan fluorescent protein (AmCyan), Zoanthus sp. green fluorescent protein (ZsGreen), Zoanthus sp. yellow fluorescent protein (ZsYellow), Discosoma sp red fluorescent protein (DsRed), Anemonia sulcata fluorescent protein (AsRed), and Heteractis crispa red fluorescent protein (HcRed).
6. The composition of claim 1 , wherein the Fc-region is derived from mouse immunoglobulin.
7. The composition of claim 1 , wherein the linker is an enzyme-cleavable linker.
8. The composition of claim 7 , wherein the enzyme that cleaves the linker is a protease.
9. The composition of claim 1 , wherein the FRET pair comprises one of CFP-YFP, GFP-mRFP1, YFP-mRFP1, or GFP-RFP.
10. The composition of claim 1 , wherein at least some of the linkers have a length of 10 nm or less.
11. A FRET method, the method comprising:
obtaining the composition of claim 1 ;
irradiating the composition with light having a wavelength and intensity sufficient to excite detectable fluorescence and FRET emission from one or both of the first and second fluorescent proteins; and
obtaining a first fluorescence signal from at least one of the first or second fluorescent proteins.
12. The composition of claim 1 , wherein the marker is a fluorophore with a characteristic emission spectrum.
13. The composition of claim 12 , wherein the fluorophore with a characteristic emission spectrum is one of the first or second fluorescent proteins.