Kim Sung-Bae, Nishihara Ryo, Suzuki Koji
Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba, 305-8569, Ibaraki, Japan.
Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama, 223-8522, Kanagawa, Japan.
Methods Mol Biol. 2016;1461:183-93. doi: 10.1007/978-1-4939-3813-1_15.
Optical imaging of protein-protein interactions (PPIs) facilitates comprehensive elucidation of intracellular molecular events. The present protocol demonstrates an optical measure for visualizing molecular tension triggered by any PPI in mammalian cells. A unique design of single-chain probes was fabricated, in which a full-length artificial luciferase (ALuc(®)) was sandwiched between two model proteins of interest, e.g., FKBP and FRB. A molecular tension probe comprising ALuc23 greatly enhances the bioluminescence in response to varying concentrations of rapamycin, and named "tension probe (TP)." The basic probe design can be further modified towards eliminating the C-terminal end of ALuc and was found to improve signal-to-background ratios, named "combinational probe." TPs may become an important addition to the tool box of bioassays in the determination of protein dynamics of interest in mammalian cells.
蛋白质-蛋白质相互作用（PPI）的光学成像有助于全面阐明细胞内分子事件。本方案展示了一种用于可视化哺乳动物细胞中任何PPI引发的分子张力的光学测量方法。构建了一种独特设计的单链探针，其中全长人工荧光素酶（ALuc®）夹在两个感兴趣的模型蛋白之间，例如FKBP和FRB。一种包含ALuc23的分子张力探针在响应不同浓度的雷帕霉素时极大地增强了生物发光，并被命名为“张力探针（TP）”。基本探针设计可进一步改进以去除ALuc的C末端，发现这可提高信噪比，命名为“组合探针”。在确定哺乳动物细胞中感兴趣的蛋白质动力学时，TPs可能成为生物测定工具箱中的重要补充。
