In Planta Visualization of Protein Interactions Using Bimolecular Fluorescence Complementation (BiFC).
In Planta Visualization of Protein Interactions Using Bimolecular Fluorescence Complementation (BiFC).
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DOI：
10.1101/pdb.prot4995
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发表时间：
2008-04-01
期刊：
影响因子：
--
通讯作者：
Kudla, Jorg
中科院分区：
文献类型：
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作者：
Waadt, Rainer;Kudla, Jorg
引言双分子荧光互补（BiFC）分析可以直接观察活细胞中的蛋白质-蛋白质相互作用。该方法已成功地适用于不同生物体中的各种表达系统。BiFC是基于增强型黄色荧光蛋白（eYFP）的片段在通过与这些片段融合的两个缔合蛋白的相互作用聚集在一起时形成荧光复合物。这些蛋白质的相互作用恢复荧光，并允许蛋白质复合物的空间定位模式的可视化。相互作用的缺乏阻止了荧光蛋白的重组，并且仅导致背景荧光。双分子荧光互补的特异性必须通过对相互作用界面发生突变的蛋白质进行平行分析来确认。该方案描述了用于本氏烟草叶细胞中BiFC测定的农杆菌介导的瞬时表达方案。该方法表现出高转化率（高达90%的细胞），并允许在单个细胞中同时表达多种蛋白质。因此，该表达系统使得荧光标记的蛋白质与BiFC复合物的形成的共定位分析成为可能，用于确定细胞复合物定位。此外，蛋白质相互作用的分析，在N。本塞姆那叶允许研究在不同表达时间点的蛋白质相互作用，允许分析在原生质体中通常有毒的蛋白质，并且使得能够在表皮细胞以及叶肉原生质体中进行比较性蛋白质相互作用研究。
INTRODUCTIONBimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein-protein interactions in living cells. This method has been successfully adapted to a variety of expression systems in different organisms. BiFC is based on the formation of a fluorescent complex by fragments of the enhanced yellow fluorescent protein (eYFP) when brought together by the interaction of two associating proteins fused to these fragments. Interaction of these proteins restores fluorescence and allows the visualization of spatial localization patterns of protein complexes. Absence of interaction prevents reassembly of the fluorescent protein and results only in background fluorescence. The specificity of bimolecular fluorescence complementation must be confirmed by parallel analysis of proteins in which the interaction interface has been mutated. This protocol describes the Agrobacterium-mediated transient expression protocol for BiFC assays in Nicotiana benthamiana leaf cells. This method exhibits a high transformation rate (up to 90% of the cells) and allows the simultaneous expression of multiple proteins in single cells. Therefore, this expression system enables colocalization analyses of fluorescently labeled proteins with the formation of BiFC complexes for determination of cellular complex localization. In addition, protein interaction assays in N. benthamiana leaves permit the investigation of protein interactions at different time points of expression, allow analysis of proteins that are normally toxic in protoplasts, and enable comparative protein interaction investigation in epidermal cells as well as in mesophyll protoplasts.