A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins.
We describe a method for assaying protein interactions that offers some attractive advantages over previous assays. This method, called bioluminescence resonance energy transfer (BRET), uses a bioluminescent luciferase that is genetically fused to one candidate protein, and a green fluorescent protein mutant fused to another protein of interest. Interactions between the two fusion proteins can bring the luciferase and green fluorescent protein close enough for resonance energy transfer to occur, thus changing the color of the bioluminescent emission. By using proteins encoded by circadian (daily) clock genes from cyanobacteria, we use the BRET technique to demonstrate that the clock protein KaiB interacts to form homodimers. BRET should be particularly useful for testing protein interactions within native cells, especially with integral membrane proteins or proteins targeted to specific organelles.
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Department of Biology, Box 1812-B, Vanderbilt University, Nashville, TN 37235, USA.
Piston
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DW
Johnson
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CH
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001683:Biological Clocks / Q000502:physiology*; D002940:Circadian Rhythm / Q000502:physiology*; D056925:Circadian Rhythm Signaling Peptides and Proteins; D003001:Cloning, Molecular; D000458:Cyanobacteria; D019281:Dimerization; D004735:Energy Transfer; D004926:Escherichia coli / Q000235:genetics; D008156:Luciferases / Q000235:genetics; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins; D013050:Spectrometry, Fluorescence / Q000379:methods*
D001426:Bacterial Proteins; D056925:Circadian Rhythm Signaling Peptides and Proteins; C114269:KaiA protein, cyanobacteria; C114270:KaiB protein, cyanobacteria; C114271:KaiC protein, cyanobacteria; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D008156:Luciferases