Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta
Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta
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DOI：
10.1016/j.jmb.2006.08.017
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发表时间：
2006-10-06
影响因子：
5.6
通讯作者：
Tzfira, Tzvi
中科院分区：
文献类型：
--
作者：
Citovsky, Vitaly;Lee, Lan-Ying;Tzfira, Tzvi
双分子荧光互补（BiFC）是研究和可视化活细胞中蛋白质相互作用的最先进和最强大的工具之一。在该方法中，将推定的相互作用蛋白质配偶体融合到自体荧光蛋白的互补非荧光片段，例如绿色荧光蛋白的黄色光谱变体。如果绿色荧光蛋白的黄色光谱变体的两个部分以它们可以适当折叠的方式结合在一起，则测试蛋白的相互作用可能导致荧光的重建。BiFC提供了一种检测蛋白质-蛋白质相互作用以及相互作用蛋白质伴侣的亚细胞定位的测定。为了促进BiFC在植物研究中的应用，我们设计了一系列载体，用于容易地构建目标蛋白与绿色荧光蛋白片段的黄色光谱变体的N-末端和C-末端融合。这些载体携带具有扩展的多克隆位点的组成型表达盒。此外，这些载体促进BiFC表达盒组装到农杆菌多基因表达二元质粒中，用于共表达相互作用伴侣和额外的自体荧光蛋白，其可用作内部转化对照和亚细胞区室的标记物。我们证明了这些载体的实用性，在不同的细胞室，包括细胞核，胞间连丝，叶绿体的不同植物物种和细胞类型的特定蛋白质蛋白质相互作用的分析。(c)2006爱思唯尔有限公司保留所有权利。
Bimolecular fluorescence complementation (BiFC) represents one of the most advanced and powerful tools for studying and visualizing proteinprotein interactions in living cells. In this method, putative interacting protein partners are fused to complementary non-fluorescent fragments of an autofluorescent protein, such as the yellow spectral variant of the green fluorescent protein. Interaction of the test proteins may result in reconstruction of fluorescence if the two portions of yellow spectral variant of the green fluorescent protein are brought together in such a way that they can fold properly. BiFC provides an assay for detection of protein-protein interactions, and for the subcellular localization of the interacting protein partners. To facilitate the application of BiFC to plant research, we designed a series of vectors for easy construction of N-terminal and C-tern-Linal fusions of the target protein to the yellow spectral variant of the green fluorescent protein fragments. These vectors carry constitutive expression cassettes with an expanded multi-cloning site. In addition, these vectors facilitate the assembly of BiFC expression cassettes into Agrobacterium multigene expression binary plasmids for co-expression of interacting partners and additional autofluorescent proteins that may serve as internal transformation controls and markers of subcellular compartments. We demonstrate the utility of these vectors for the analysis of specific proteinprotein interactions in various cellular compartments, including the nucleus, plasmodesmata, and chloroplasts of different plant species and cell types. (c) 2006 Elsevier Ltd. All rights reserved.