Direct comparison of bioluminescence-based resonance energy transfer methods for monitoring of proteolytic cleavage

Bioluminescence resonance energy transfer (BRET) is a powerful tool for the study of protein-protein interactions and conformational changes within proteins. Two common implementations of BRET are BRET1 with Renilla luciferase (RLuc) and coelenterazine It (CLZ, lambda(em) similar to 475 nm) and BRET2 with the substrate coelenterazine 400a (CLZ400A substrate, lambda(em) = 395 nm) as the respective donors. For BRET1 the acceptor is yellow fluorescent protein (YFP) (lambda(em) similar to 535 nm), a mutant of green fluorescent protein (GFP), and for BRET2 it is GFP(2) (lambda(em) similar to 515 nm). It is not clear from previous studies which of these systems has superior signal-to-background characteristics. Here we directly compared BRET1 and BRET2 by placing two different protease-specific cleavage sequences between the donor and acceptor domains. The intact proteins simulate protein-protein association. Proteolytic cleavage of the peptide linker simulates protein dissociation and can be detected as a change in the BRET ratios. Complete cleavage of its target sequence by thrombin changed the BRET2 ratio by a factor of 28.9 +/- 0.2 (relative standard deviation [RSD], n = 3) and changed the BRET1 ratio by a factor of 3.05 +/- 0.07. Complete cleavage of a caspase-3 target sequence resulted in the BRET ratio changes by factors of 15.45 +/- 0.08 for BRET2 and 2.00 +/- 0.04 for BRET1. The BRET2 assay for thrombin was 2.9 times more sensitive compared with the BRET1 version. Calculated detection limits (blank signal + 3 sigma(b), where sigma(b) = standard deviation [SD] of blank signal) were 53 pM (0.002 U) thrombin with BRET1 and 15 pM (0.0005 U) thrombin with BRET2. The results presented here suggest that BRET2 is a more suitable system than BRET1 for studying protein-protein interactions and as a potential sensor for monitoring Protease activity. Crown Copyright (C) 2008 Published by Elsevier Inc. All rights reserved.