BRET3: a red-shifted bioluminescence resonance energy transfer (BRET)-based integrated platform for imaging protein-protein interactions from single live cells and living animals.
Taking advantage of the bioluminescence resonance energy transfer (BRET) phenomenon, we report the development of a highly photon-efficient, self-illuminating fusion protein combining a mutant red fluorescent protein (mOrange) and a mutant Renilla reniformis luciferase (RLuc8). This new BRET fusion protein (BRET3) exhibits severalfold improvement in light intensity in comparison with existing BRET fusion proteins. BRET3 also exhibits the most red-shifted light output (564-nm peak wavelength) of any reported bioluminescent protein that utilizes its natural substrate coelenterazine, a benefit of which is demonstrated at various tissue depths in small animals. The imaging utility of BRET3 at the single-cell level is demonstrated using an intramolecular sensor incorporating two mammalian target of rapamycin pathway proteins (FKBP12 and FRB) that dimerize only in the presence of rapamycin. With its increased photon intensity, red-shifted light output, and good spectral resolution (approximately 85 nm), BRET3 shows improved spatial and temporal resolution for measuring intracellular events in single cells and in living small animal models. The development of further BRET3-based assays will allow imaging of protein-protein interactions using a single assay directly scalable from intact living cells to small living subjects, allowing accelerated drug discovery.
Stanford University School of Medicine, Department of Radiology, Bioengineering and Bio-X Program, The James H. Clark Center, 318 Campus Dr., Stanford, CA 94305-5427, USA.
Abhijit
A
De
Pritha
P
Ray
Andreas Markus
AM
Loening
Sanjiv Sam
SS
Gambhir
D000818:Animals; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D049410:Luciferases, Renilla / Q000235:genetics / Q000378:metabolism; D049449:Luminescence; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D022061:Tacrolimus Binding Protein 1A / Q000378:metabolism; D000097573:Red Fluorescent Protein
D049410:Luciferases, Renilla; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D022061:Tacrolimus Binding Protein 1A; D000097573:Red Fluorescent Protein; C413663:fluorescent protein 583