Waadt Rainer, Kudla Jörg
Institut für Botanik und Botanischer Garten, Universität Münster, 48149 Münster, Germany.
CSH Protoc. 2008 Apr 1;2008:pdb.prot4995. doi: 10.1101/pdb.prot4995.
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.
引言
双分子荧光互补（BiFC）分析能够直接观察活细胞中的蛋白质-蛋白质相互作用。该方法已成功应用于不同生物体的多种表达系统。BiFC基于当与这些片段融合的两个相互作用蛋白相互作用时，增强型黄色荧光蛋白（eYFP）片段形成荧光复合物。这些蛋白质的相互作用恢复荧光，并允许观察蛋白质复合物的空间定位模式。缺乏相互作用会阻止荧光蛋白的重新组装，仅产生背景荧光。双分子荧光互补的特异性必须通过对相互作用界面已发生突变的蛋白质进行平行分析来确认。本方案描述了在本氏烟草叶细胞中进行BiFC分析的农杆菌介导的瞬时表达方案。该方法具有高转化率（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，该表达系统能够对荧光标记的蛋白质进行共定位分析，并形成BiFC复合物以确定细胞复合物的定位。此外，在本氏烟草叶片中进行蛋白质相互作用分析，可以在表达的不同时间点研究蛋白质相互作用，分析在原生质体中通常有毒的蛋白质，并能够在表皮细胞以及叶肉原生质体中进行比较蛋白质相互作用研究。
