Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan.
Biotechniques. 2012 Jan;52(1):45-50. doi: 10.2144/000113777.
The bimolecular fluorescence complementation (BiFC) assay is a method for visualizing protein-protein interactions in living cells. To visualize the cofilin-actin interaction in living cells, a series of combinations of the N- and C-terminal fragments of Venus fused upstream or downstream of cofilin and actin were screened systematically. A new pair of split Venus fragments, Venus (1-210) fused upstream of cofilin and Venus (210-238) fused downstream of actin, was the most effective combination for visualizing the specific interaction between cofilin and actin in living cells. This pair of Venus fragments was also effective for detecting the active Ras-dependent interaction between H-Ras and Raf1 and the Ca(2+)-dependent interaction between calmodulin and its target M13 peptide. In vitro BiFC assays using the pair of purified BiFC probes provided the means to detect the specific interactions between cofilin and actin and between H-Ras and Raf1. In vivo and in vitro BiFC assays using the newly identified pair of Venus fragments will serve as a useful tool for measuring protein-protein interactions with high specificity and low background fluorescence and could be applied to the screening of inhibitors that block protein-protein interactions.
双分子荧光互补（BiFC）测定法是一种用于在活细胞中可视化蛋白质-蛋白质相互作用的方法。为了在活细胞中可视化肌动蛋白与丝切蛋白的相互作用，我们系统地筛选了一系列将 Venus 的 N-和 C-末端片段融合到丝切蛋白和肌动蛋白的上游或下游的组合。由 Venus（1-210）融合到丝切蛋白的上游和 Venus（210-238）融合到肌动蛋白的下游组成的新的一对 Venus 片段，是用于在活细胞中可视化丝切蛋白与肌动蛋白之间特异性相互作用的最有效组合。该对 Venus 片段也可用于检测 H-Ras 与 Raf1 之间的活性 Ras 依赖性相互作用以及钙调蛋白与其靶标 M13 肽之间的 Ca2+依赖性相互作用。使用纯化的 BiFC 探针进行的体外 BiFC 测定法为检测丝切蛋白与肌动蛋白之间以及 H-Ras 与 Raf1 之间的特异性相互作用提供了手段。使用新鉴定的 Venus 片段进行的体内和体外 BiFC 测定法将成为一种有用的工具，用于测量具有高特异性和低背景荧光的蛋白质-蛋白质相互作用，并且可以应用于筛选阻止蛋白质-蛋白质相互作用的抑制剂。
