科研之友
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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 of BRET are BRET(1) with Renilla luciferase (RLuc) and coelenterazine It (CLZ, lambda(em) similar to 475 nm) and BRET(2) with the substrate coelenterazine 400a (CLZ400A substrate, lambda(em) = 395 nm) as the respective donors. For BRET(1) the acceptor is yellow fluorescent protein (YFP) (lambda(em) similar to 535 nm), a mutant of green fluorescent protein (GFP), and for BRET(2) 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 BRET(1) and BRET(2) 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 BRET(2) ratio by a factor of 28.9 /- 0.2 (relative standard deviation [RSD], n = 3) and changed the BRET(1) 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 BRET(2) and 2.00 /- 0.04 for BRET(1). The BRET(2) assay for thrombin was 2.9 times more sensitive compared with the BRET(1) 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 BRET(1) and 15 pM (0.0005 U) thrombin with BRET(2). The results presented here suggest that BRET(2) is a more suitable system than BRET(1) for studying protein-protein interactions and as a potential sensor for monitoring Protease activity.
- 出版日期2009-2-15
- 单位CSIRO
