Woo Jongchan, Hong Jason, Dinesh-Kumar Savithramma P
Department of Plant Biology and the Genome Center, College of Biological Sciences, University of California, Davis, California.
Curr Protoc Protein Sci. 2017 Apr 3;88:19.30.1-19.30.12. doi: 10.1002/cpps.30.
Bioluminescence resonance energy transfer (BRET) is a technique that analyzes protein-protein interactions (PPIs). The unique feature of BRET delineates that the resonance energy is generated by the resonance energy donor, Renilla luciferase by the oxidative decarboxylation of coelenterazine substrate. BRET is superior to FRET where issues such as autofluorescence, photobleaching, and light scattering can occur. Recently, BRET has been applied to design synthetic biosensors for monitoring autophagy in vivo and in vitro. Here, we report the methods for constructing a biosensor of human HsLC3a as a probe for autophagy biogenesis and the optimization of the intramolecular BRET assay that allows for high-throughput screening of chemical modulators of autophagy. User-friendly working interface with the BRET-based synthetic sensor of HsLC3a makes drug discovery easy and amenable for high-throughput. The BRET protocol described here could be easily applicable to generate other biosensors for monitoring PPIs by measurement of intermolecular BRET. © 2017 by John Wiley & Sons, Inc.
生物发光共振能量转移（BRET）是一种分析蛋白质-蛋白质相互作用（PPI）的技术。BRET的独特之处在于，共振能量由海肾荧光素酶这种共振能量供体通过腔肠素底物的氧化脱羧作用产生。BRET优于荧光共振能量转移（FRET），后者可能会出现诸如自发荧光、光漂白和光散射等问题。最近，BRET已被应用于设计用于在体内和体外监测自噬的合成生物传感器。在此，我们报告构建作为自噬生物发生探针的人HsLC3a生物传感器的方法，以及分子内BRET检测的优化，该检测允许对自噬化学调节剂进行高通量筛选。HsLC3a基于BRET的合成传感器的用户友好工作界面使药物发现变得容易且适合高通量。本文所述的BRET方案可轻松应用于通过测量分子间BRET来生成用于监测PPI的其他生物传感器。© 2017约翰威立国际出版公司
