A suite of tools and application notes for in vivo protein interaction assays using bioluminescence resonance energy transfer (BRET)
A suite of tools and application notes for in vivo protein interaction assays using bioluminescence resonance energy transfer (BRET)
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DOI：
10.1111/j.1365-313x.2006.02851.x
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发表时间：
2006-10-01
期刊：
影响因子：
7.2
通讯作者：
von Arnim, Albrecht G.
中科院分区：
文献类型：
--
作者：
Subramanian, Chitra;Woo, Jongchan;von Arnim, Albrecht G.
生物发光共振能量转移（BRET）是一种自然的生物物理现象，是一种真实的实时监测活细胞中蛋白质相互作用的新兴技术的基础。在这里，我们提出了一系列的技术进步，以提高BRET测定在植物中的实用性。产生了一系列重组克隆载体，以在瞬时测定条件下和在稳定的转基因植物中加速用海肾荧光素酶或黄色荧光蛋白标记的蛋白质的表达。在稳定转化的拟南芥或烟草中工作，然后我们检测到三对候选相互作用伙伴之间的BRET：E3泛素连接酶COP 1的二聚化，COP 1和B盒蛋白STH之间的相互作用，以及光调节bZip转录因子HY 5和HYH之间的相互作用。海肾荧光素酶基因的密码子优化版本导致拟南芥中表达的改善。海肾荧光素酶在多种亚细胞器中具有活性，包括质体、线粒体、过氧化物酶体和高尔基体。在拟南芥光信号机制作为模型系统的调查中，我们估计了已知蛋白质-蛋白质相互作用可以使用BRET记录的可能性。最后，我们表明，海肾荧光素酶可以作为一个记者的蛋白质稳定性的放线菌酮追逐试验。
Bioluminescence resonance energy transfer (BRET) is a natural biophysical phenomenon that underlies an emerging technique to monitor protein - protein interactions in living cells in real time. Here, we present a series of technical advances to enhance the utility of the BRET assay in plants. A series of recombination cloning vectors was generated to accelerate the expression of proteins tagged with Renilla luciferase or yellow fluorescent protein under transient assay conditions and in stable transgenic plants. Working in stably transformed Arabidopsis or tobacco, we then detected BRET between three pairs of candidate interaction partners: dimerization of the E3 ubiquitin ligase COP1, interaction between COP1 and the B-box protein STH, and interaction between the light regulatory bZip transcription factors HY5 and HYH. A codon- optimized version of the Renilla luciferase gene resulted in improved expression in Arabidopsis. Renilla luciferase was active in a variety of subcellular organelles, including plastids, mitochondria, peroxisomes and Golgi stacks. In a survey of the Arabidopsis light signaling machinery as a model system, we estimated the likelihood that a known protein - protein interaction can be documented using BRET. Finally, we show that Renilla luciferase may serve as a reporter of protein stability in a cycloheximide chase assay.