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

摘要: Bioluminescence resonance energy transfer (BRET) is a powerful tool for the study of protein-protein interactions and conformational changes within proteins. Two common implementations BRET are BRET(1) with Renilla luciferase (RLuc) coelenterazine h (CLZ, lambda(em) approximately 475 nm) BRET(2) substrate 400a (CLZ400A substrate, lambda(em)=395 as respective donors. For acceptor yellow fluorescent protein (YFP) (lambda(em) 535 nm), mutant green (GFP), it GFP(2) 515 nm). It not clear from previous studies which these systems has superior signal-to-background characteristics. Here we directly compared by placing two different protease-specific cleavage sequences between donor domains. The intact proteins simulate association. Proteolytic peptide linker simulates dissociation can be detected change in ratios. Complete its target sequence thrombin changed ratio factor 28.9+/-0.2 (relative standard deviation [RSD], n=3) 3.05+/-0.07. caspase-3 resulted factors 15.45+/-0.08 2.00+/-0.04 BRET(1). assay was 2.9 times more sensitive version. Calculated detection limits (blank signal+3sigma(b), where sigma(b)=standard [SD] blank signal) were 53 pM (0.002 U) 15 (0.0005 BRET(2). results presented here suggest that suitable system than studying potential sensor monitoring protease activity.