Multiplexing of Multicolor Bioluminescence Resonance Energy Transfer
Multiplexing of Multicolor Bioluminescence Resonance Energy Transfer
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DOI：
10.1016/j.bpj.2010.10.025
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发表时间：
2010-12-15
影响因子：
3.4
通讯作者：
Bouvier, Michel
中科院分区：
文献类型：
--
作者：
Breton, Billy;Sauvageau, Etienne;Bouvier, Michel
生物发光共振能量转移（BRET）越来越多地被用于监测蛋白质-蛋白质相互作用和细胞中的细胞事件。然而，同时监测多个事件的能力受到现有BRET伙伴光谱特性的限制。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FPs创建新型BRET构型的可能性。产生并验证了三种新的（据我们所知）BRET测定，导致不同颜色的生物发光发射。使用的两种FP（增强蓝（EB）FP 2和mAmetrine）的光谱特性和选择适当的检测滤波器允许在添加海肾酶-II的独特底物coelentrazine-400 a后在相同细胞中同时检测两个独立的BRET信号，而没有交叉干扰。使用基于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蛋白沿着第二信使的产生的活化，我们建立了两种新BRET构型可以复用以同时监测两种依赖或独立的细胞事件的原理证明。这种新的多重BRET配置的发展开辟了同时监测活细胞中各种独立的生物过程的途径。
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.