Bioluminescence Resonance Energy Transfer (BRET)-based Biosensing Probes Using Novel Luminescent and Fluorescent Protein Pairs

Bioluminescence resonance energy transfer (BRET) is a useful technique for visualizing cellular functions and responses to stimuli. To construct efficient biosensing protein probes using BRET, novel luminescent and red fluorescent protein pairs, which have separate peaks of luminescence and fluorescence and can cause energy transfer efficiently, were screened. The red fluorescent protein, mScarletI, used as an acceptor, induced a highly efficient BRET signal from green or blue luminescent proteins [Emerald Luc (ELuc) or NanoLuc (NLuc)]. Novel pairs of luminescent and red fluorescent protein (mScarletI) could be applied to the analysis of calcium ions (Ca 2+ ). The BRET-based biosensing protein pair of mScarletI and NLuc showed an increased intensity of the BRET signal, depending on the concentration of Ca 2+ (0-4 M). Intracellular Ca 2+ influx was monitored in HEK293A cells stimulated with 50 mM KCl and 15 mM arginine using the BRET-based biosensing protein probe with the novel protein pair. This pair of proteins was particularly suited to cellular imaging in vitro and even in vivo. Therefore, it could be useful for various BRET-based analyses of cell and tissue samples.