Department of Biochemistry, Institute for Research in Immunology and Cancer, and Groupe de Recherche Universitaire sur le Médicament, Université de Montréal, Montréal, Canada.
Biophys J. 2010 Dec 15;99(12):4037-46. doi: 10.1016/j.bpj.2010.10.025.
Bioluminescence resonance energy transfer (BRET) is increasingly being used to monitor protein-protein interactions and cellular events in cells. However, the ability to monitor multiple events simultaneously is limited by the spectral properties of the existing BRET partners. Taking advantage of newly developed Renilla luciferases and blue-shifted fluorescent proteins (FPs), we explored the possibility of creating novel BRET configurations using a single luciferase substrate and distinct FPs. Three new (to our knowledge) BRET assays leading to distinct color bioluminescence emission were generated and validated. The spectral properties of two of the FPs used (enhanced blue (EB) FP2 and mAmetrine) and the selection of appropriate detection filters permitted the concomitant detection of two independent BRET signals, without cross-interference, in the same cells after addition of a unique substrate for Renilla luciferase-II, coelentrazine-400a. Using individual BRET-based biosensors to monitor the interaction between G-protein-coupled receptors and G-protein subunits or activation of different G-proteins along with the production of a second messenger, we established the proof of principle that two new BRET configurations can be multiplexed to simultaneously monitor two dependent or independent cellular events. The development of this new multiplexed BRET configuration opens the way for concomitant monitoring of various independent biological processes in living cells.
生物发光共振能量转移（BRET）技术越来越多地被用于监测细胞内的蛋白质-蛋白质相互作用和细胞事件。然而，由于现有 BRET 伴侣的光谱特性，同时监测多个事件的能力受到限制。利用新开发的海肾荧光素酶和蓝移荧光蛋白（FPs），我们探索了使用单一荧光素酶底物和不同的 FPs 创建新型 BRET 构型的可能性。生成并验证了三种导致不同颜色生物发光发射的新（据我们所知）BRET 测定法。所使用的两种 FPs（增强型蓝色（EB）FP2 和 mAmetrine）的光谱特性以及适当检测滤波器的选择，允许在添加海肾荧光素酶-II 的独特底物 coelentrazine-400a 后，在同一细胞中同时检测两个独立的 BRET 信号，而不会发生交叉干扰。使用单个基于 BRET 的生物传感器来监测 G 蛋白偶联受体和 G 蛋白亚基之间的相互作用或不同 G 蛋白的激活以及第二信使的产生，我们确立了两个新的 BRET 构型可以被复用以同时监测两个依赖或独立的细胞事件的原理证明。这种新的复用 BRET 配置的开发为在活细胞中同时监测各种独立的生物过程开辟了道路。
