PDF(Pubmed)
Abstract：
The experimental identification of protein-protein interactions (PPIs) is critical to understand protein function. Thus, a plethora of sensitive and versatile approaches have been developed to detect PPIs in vitro or in vivo, such as protein pull-down, yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP), and bimolecular fluorescence complementation (BiFC) assays. The recently established split-luciferase complementation (Split-LUC) imaging assay has several advantages compared to other approaches to detect PPIs in planta: it is a relatively simple and fast method to detect PPIs in vivo; the results are quantitative, with high sensitivity and low background; it measures dynamic PPIs in real-time; and it requires limited experimental materials and instrumentation. In this assay, the amino-terminal and carboxyl-terminal halves of the luciferase enzyme are fused to two proteins of interest (POIs), respectively; the luciferase protein is reconstituted when two POIs interact with each other, giving rise to a measurable activity. Here, we describe a protocol for the Split-LUC imaging assay using a pair of modified gateway-compatible vectors upon Agrobacterium-mediated transient expression in Nicotiana benthamiana. With this setup, we have successfully confirmed a series of interactions among virus-plant proteins, virus-virus proteins, plant-plant proteins, or bacteria-plant proteins in N. benthamiana.
摘要：
蛋白质-蛋白质相互作用（PPI）的实验鉴定对于理解蛋白质功能至关重要。因此，已经开发了许多灵敏和通用的方法来检测体外或体内的PPI，比如蛋白质下拉，酵母双杂交（Y2H），免疫共沉淀（co-IP），和双分子荧光互补(BiFC)测定。最近建立的分裂荧光素酶互补（Split-LUC）成像测定与其他检测植物中PPI的方法相比具有几个优点：它是一种在体内检测PPI的相对简单和快速的方法；结果是定量的，具有高灵敏度和低背景;它实时测量动态PPI;它需要有限的实验材料和仪器。在这个试验中,荧光素酶的氨基末端和羧基末端的一半与两种感兴趣的蛋白质（POI）融合，当两个POI相互作用时，荧光素酶蛋白被重建，产生可衡量的活动。这里,我们描述了Split-LUC成像分析的方案,该方案使用一对改良的网关兼容载体对农杆菌介导的烟草瞬时表达进行分析.有了这个设置，我们已经成功地证实了病毒-植物蛋白之间的一系列相互作用，病毒-病毒蛋白，植物-植物蛋白，或者是N.benthamiana中的细菌植物蛋白。
