Kim Hee-Yeon, Lee Joong-jae, Kim Nury, Heo Won Do, Kim Hak-Sung
Graduate School of Nanoscience and Technology (WCU), Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701, South Korea.
Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701, South Korea.
Biochem Biophys Res Commun. 2016 Feb 19;470(4):857-63. doi: 10.1016/j.bbrc.2016.01.129. Epub 2016 Jan 26.
Probing protein-protein interactions in living cells is crucial for understanding the protein functions and developing drugs. Small-sized protein binders are considered effective and useful for such analysis. Here we describe the development and use of a repebody, which is a protein binder composed of LRR (Leucine-rich repeat) modules, for tracking protein-protein interaction and localization in real-time through live-cell imaging. A repebody with high affinity for a red fluorescent protein was selected through a phage display, fused with a green fluorescent protein, and applied for tracing a red fluorescent protein-fused target protein in mammalian cells. The potential and utility of our approach was demonstrated by tracking the rapamycin-mediated interaction between FKBP12-rapamycin binding (FRB) domain and a FK506-binding protein (FKBP) and their localization by live-cell imaging. The present approach can be widely used for the analysis of protein-protein interaction and an understanding of complex biological processes in living cells.
在活细胞中探究蛋白质-蛋白质相互作用对于理解蛋白质功能和开发药物至关重要。小型蛋白质结合剂被认为对这种分析有效且有用。在此，我们描述了一种重复体的开发和应用，它是一种由富含亮氨酸重复序列（LRR）模块组成的蛋白质结合剂，用于通过活细胞成像实时追踪蛋白质-蛋白质相互作用和定位。通过噬菌体展示筛选出对红色荧光蛋白具有高亲和力的重复体，将其与绿色荧光蛋白融合，并用于在哺乳动物细胞中追踪红色荧光蛋白融合的靶蛋白。通过活细胞成像追踪雷帕霉素介导的FKBP12-雷帕霉素结合（FRB）结构域与FK506结合蛋白（FKBP）之间的相互作用及其定位，证明了我们方法的潜力和实用性。本方法可广泛用于蛋白质-蛋白质相互作用分析以及理解活细胞中的复杂生物学过程。
