Comparison of enhanced bioluminescence energy transfer donors for protease biosensors

Bioluminescence energy transfer (BRET) is a powerful tool for the study of protein-protein interactions and conformational changes within proteins. We directly compared two recently developed variants of Renilla luciferase (RLuc), RLuc2 and RLuc8, as BRET donors using an in vitro thrombin assay. The comparison was carried out by placing a thrombin-specific cleavage sequence between the donor luciferase and a green fluorescent protein (GFP2) acceptor. Substitution of native RLuc with the RLuc mutants, RLuc2 and 8, in a BRET2 fusion protein increased the light output by a factor of ∼10. Substitution of native RLuc with either of the RLuc mutants resulted in a decrease in BRET2 ratio by a factor of ∼2 when BRET2 components were separated by the thrombin cleavage sequence. BRET2 ratios changed by factors of 18.8 ± 1.2 and 18.2 ± 0.4 for GFP2-RG-RLuc2 and GFP2-RG-RLuc8 fusion proteins, respectively, on thrombin cleavage compared to 28.8 ± 0.20 for GFP2-RG-RLuc. The detection limits for thrombin were 0.23 and 0.26 nM for RLuc2 and RLuc8 BRET2 systems, respectively, and 15 pM for GFP2-RG-RLuc. However, overall, the mutant BRET systems remain more sensitive than FRET and brighter than standard BRET2.