INSERM UMR1037, Cancer Research Center of Toulouse, Université de Toulouse, Institut Claudius Regaud, F-31052 Toulouse, France.
Sci Rep. 2013 Oct 4;3:2854. doi: 10.1038/srep02854.
Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence.
监测活细胞中的蛋白质-蛋白质相互作用对于揭示它们在许多细胞过程和各种疾病中的作用至关重要。以前描述的基于 GFP 分裂的传感器存在折叠和/或自组装背景荧光不佳的问题。在这里，我们设计了一种微标记系统来监测体内和体外的蛋白质-蛋白质相互作用。该测定基于三个部分的关联，即两个二十个氨基酸长的 GFP 标签 GFP10 和 GFP11 与相互作用的蛋白质伴侣融合，以及互补的 GFP1-9 检测物。当蛋白质相互作用时，GFP10 和 GFP11 与 GFP1-9 自组装以重新构成功能性 GFP。我们使用卷曲螺旋和 FRB/FKBP12 模型系统在体外和大肠杆菌中对传感器进行了表征。我们将研究扩展到哺乳动物细胞，并检查 FK-506 对 rapamycin 诱导的 FRB/FKBP12 结合的抑制作用。这些标签的体积小，对融合蛋白行为和溶解度的影响最小，应该能够通过荧光监测蛋白质-蛋白质的结合来开展新的实验。
