Protein–protein interaction is one of the key problems in mechanistic studies of critical cellular processes and diseases. Fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) are among a range of available methods for studies on such interaction. In brief, the resonance energy transfer refers to energy transfer between fluorescent/luminescent donor and acceptor molecules when they are in close proximity (1–10 nm) with properly oriented dipoles. Frequently used donors include blue fluorescent protein (BFP) and cyan fluorescent protein (CFP) for FRET, and luminescent products of luciferase for BRET. Commonly used acceptors include GFP, Venus, Citrine, YPet and mOrange. Efficiency of transfer and resolution of detection are greatly influenced by spectral properties of both donors and acceptors, in addition to expression ratio of donor and acceptor fusion proteins21. Donor–acceptor pairs with compatible spectral properties ensure efficient energy transfer and high signal to noise ratio. The emission spectrum of a donor must to some extent overlap the excitation spectrum of the acceptor to enable high efficient transfer, but a pronounced overlap would, on the other hand, sacrifice detection resolution resulting in poor signal to noise ratio. Moreover, efficiency is also markedly affected by comparative expression of donor vs. acceptor fusion proteins, especially when they form homogeneous dimmers21. Thus, it is critical to optimize protein expression pattern before prior to HTS campaign.