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 (GFP(2)) acceptor. Substitution of native RLuc with the RLuc mutants, RLuc2 and 8, in a BRET(2) 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 BRET(2) ratio by a factor of ~2 when BRET(2) components were separated by the thrombin cleavage sequence. BRET(2) ratios changed by factors of 18.8±1.2 and 18.2±0.4 for GFP(2)-RG-RLuc2 and GFP(2)-RG-RLuc8 fusion proteins, respectively, on thrombin cleavage compared to 28.8±0.20 for GFP(2)-RG-RLuc. The detection limits for thrombin were 0.23 and 0.26 nM for RLuc2 and RLuc8 BRET(2) systems, respectively, and 15 pM for GFP(2)-RG-RLuc. However, overall, the mutant BRET systems remain more sensitive than FRET and brighter than standard BRET(2).