Detection of Protein-Protein Interactions in Bacteria by GFP-Fragment Reconstitution
Detection of Protein-Protein Interactions in Bacteria by GFP-Fragment Reconstitution
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DOI：
10.1007/978-1-61737-967-3_15
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发表时间：
2011-01-01
期刊：
影响因子：
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通讯作者：
Umezawa, Yoshio
中科院分区：
文献类型：
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作者：
Kanno, Akira;Ozawa, Takeaki;Umezawa, Yoshio
蛋白质-蛋白质相互作用是蛋白质在生物体中最关键的作用之一。蛋白质的结合/解离反映了对信号通路内在或外在扰动的反应，涉及基因表达、细胞分裂、细胞分化和凋亡。为了进一步了解生物过程，监测模型生物中蛋白质-蛋白质相互作用非常重要。特别是，基于大肠杆菌的方法适合评估大型蛋白质文库。由于毒性或表达不良，许多这些蛋白质不能在酵母中使用。在此，我们描述了一种基于内含肽介导的蛋白质重构系统（PRS）的通用方法，用于检测细菌细胞中蛋白质-蛋白质相互作用。基于 PRS 的方法不需要其他试剂，包括酶、底物和 ATP。另一个优点是成熟的绿色荧光蛋白 (GFP) 在目标细胞中积累直至降解。这些可以高度灵敏地筛选蛋白质-蛋白质相互作用。
Protein-protein interaction is one of the most pivotal roles of proteins in living organisms. Association/dissociation of proteins reflects responses to intrinsic or extrinsic perturbations of signaling pathways, involved in gene expression, cell division, cell differentiation, and apoptosis. For further understanding of the biological processes, it is important to monitor protein-protein interactions in model organisms. In particular, Escherichia coli-based methods are suitable to assess large libraries of proteins. Many of these proteins cannot be used in yeast due to toxicity or poor expression. Herein we describe a general method based on an intein-mediated protein reconstitution system (PRS) to detect protein-protein interactions in bacterial cells. The PRS-based approach requires no other agents including enzymes, substrates, and ATP. Another advantage is that matured green fluorescent protein (GFP) accumulates in a targeted cell till degraded. These allow highly sensitive screening of protein-protein interactions.