Engineering Aspects of Bioluminescence Resonance Energy Transfer Systems

Engineering BRET-Sensor Proteins

“…Until recently, the most popular luciferases were firefly luciferase and Renilla luciferase (De et al, 2014;Saito & Nagai, 2015). Firefly luciferase is a 61-kDa protein that emits orange light (λ max ¼ 562 nm) by oxidation of D-luciferin, which requires ATP as a cosubstrate.…”

“…Finally, fluorescence is ill-suited for use in optogenetics or cells that are endogenously light sensitive. In principle, all of these issues can be resolved by using sensors based on bioluminescence resonance energy transfer (BRET) (Bacart, Corbel, Jockers, Bach, & Couturier, 2008;De, Arora, & Jasani, 2014;Machleidt et al, 2015;Paley & Prescher, 2014). In these sensors, the donor fluorescent domain is replaced by a luciferase enzyme, which generates light by catalyzing the oxidation of specific organic substrate molecules.…”

Novel BRET combination for detection of rapamycin-induced protein dimerization using luciferase from fungus Neonothopanus nambi

“…FRET and BRET require two or more photoactive molecules — a donor and an acceptor situated at a specific orientation and interacting with each other at a very short distance (<10 nm). Resonance energy transfer from the excited donor to the acceptor molecule, which emits light at its characteristic wavelength [ 4 , 5 ]. Approaches based on BRET are favored over FRET ones since they do not require the external light for excitation of the donor molecule, and the luciferin-luciferase reaction is the sole source of energy transfer to the fluorophore.…”

In Vivo Biosensing Using Resonance Energy Transfer

“…BRET3 using Rluc8/mOrange BRET-pair with coelenterazine as the substrate exhibits red-shift light emission for improved imaging in animal models and several-fold better light emission compared to BRET1 and BRET2 [34]. Numerous additional BRET pairs have been demonstrated to continue to improve the photophysical properties of the protein fusions for enhanced sensitivity and wavelength tuning [35].…”