Direct comparison of bioluminescence-based resonance energy transfer methods for monitoring of proteolytic cleavage
Direct comparison of bioluminescence-based resonance energy transfer methods for monitoring of proteolytic cleavage
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DOI：
10.1016/j.ab.2008.10.040
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发表时间：
2009-02-15
影响因子：
2.9
通讯作者：
Trowell, Stephen C.
中科院分区：
文献类型：
--
作者：
Dacres, Helen;Dumancic, Mira M.;Trowell, Stephen C.
生物发光共振能量转移（BRET）是研究蛋白质-蛋白质相互作用和蛋白质内部构象变化的强大工具。 BRET 的两种常见实现是使用海肾荧光素酶 (RLuc) 和腔肠素 It（CLZ，lambda(em) 类似于 475 nm）的 BRET1 和使用底物腔肠素 400a（CLZ400A 底物，lambda(em) = 395 nm）作为各自供体的 BRET2。对于 BRET1，受体是黄色荧光蛋白 (YFP)（类似于 535 nm 的 lambda(em)），它是绿色荧光蛋白 (GFP) 的突变体；对于 BRET2，受体是 GFP(2)（类似于 515 nm 的 lambda(em)）。从之前的研究来看，尚不清楚这些系统中哪一个具有优越的信号背景特性。在这里，我们通过在供体结构域和受体结构域之间放置两个不同的蛋白酶特异性切割序列来直接比较 BRET1 和 BRET2。完整的蛋白质模拟蛋白质-蛋白质的结合。肽接头的蛋白水解切割模拟蛋白质解离，并且可以通过 BRET 比率的变化进行检测。凝血酶对其靶序列的完全切割使 BRET2 比率改变了 28.9 +/- 0.2 倍（相对标准偏差 [RSD]，n = 3），并使 BRET1 比率改变了 3.05 +/- 0.07 倍。 caspase-3 靶序列的完全切割导致 BRET2 的 BRET 比率变化为 15.45 +/- 0.08，BRET1 的 BRET 比率变化为 2.00 +/- 0.04。与 BRET1 版本相比，BRET2 凝血酶检测灵敏度高 2.9 倍。计算的检测限（空白信号 + 3 sigma(b)，其中 sigma(b) = 空白信号的标准差 [SD]）为 BRET1 的凝血酶为 53 pM (0.002 U)，BRET2 的凝血酶为 15 pM (0.0005 U)。这里提出的结果表明，BRET2 是比 BRET1 更适合研究蛋白质-蛋白质相互作用的系统，并作为监测蛋白酶活性的潜在传感器。 Crown 版权所有 (C) 2008 由 Elsevier Inc. 出版。保留所有权利。
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.