Direct comparison of fluorescence- and bioluminescence-based resonance energy transfer methods for real-time monitoring of thrombin-catalysed proteolytic cleavage

Abstract

In this study, a representative FRET system (CFP donor and YFP acceptor) is compared with the BRET 2 system ( Renilla luciferase donor, green fluorescent protein 2 (GFP 2) acceptor and coelenterazine 400a substrate). Cleavage of a thrombin-protease-sensitive peptide sequence inserted between the donor and acceptor proteins was detected by the RET signal. Complete cleavage by thrombin changed the BRET 2 signal by a factor of 28.9 ± 0.2 (R.S.D. (relative standard deviation), n = 3) and the FRET signal by a factor of 3.2 ± 0.1 (R.S.D., n = 3). The BRET 2 technique was 50 times more sensitive than the FRET technique for monitoring thrombin concentrations. Detection limits (blank signal + 3 σ b, where σ b = the standard deviation (S.D.) of the blank signal) were calculated to be 3.05 and 0.22 nM thrombin for FRET and BRET 2, respectively. This direct comparison suggests that the BRET 2 technique is more suitable than FRET for use in proximity assays such as protease cleavage assays or protein–protein interaction assays.