Rapid functional analysis of protein–protein interactions by fluorescent C‐terminal labeling and single‐molecule imaging

FEBS Letters

Sasaki, Toru¹  Tokumasu, Ako¹  Mimori-Kiyosue, Yuko³  Yamaguchi, Junichi⁴  Nemoto, Naoto¹  Yagi, Toshiki²  Funatsu, Takashi⁴ 

[1] GenCom Corporation, 11 Minamiooya, Machida, Tokyo 194-8511, Japan;Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan;Tsukita Cell Axis Project, Exploratory Research for Advanced Technology, Japan Science and Technology Corporation, Kyoto Research Park, Shimogyo-ku, Kyoto 600-8813, Japan;Department of Physics, School of Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan

关键词: Protein–protein interaction;    Single-molecule imaging;    Protein labeling;    Puromycin;    Fluorescence microscopy;    Cell-free translation;    Cy5-puro;    Cy5-conjugated puromycin;    Cy5-puro-kinesin;    kinesin labeled with Cy5-puro;    GFP;    green fluorescent protein;    AMP-PNP;    adenosine 5′-(β;    γ-imido) triphosphate;   

DOI  :  10.1016/S0014-5793(01)02581-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Detection of protein–protein interactions is a fundamental step to understanding gene function. Here we report a sensitive and rapid method for assaying protein–protein interactions at the single-molecule level. Protein molecules were synthesized in a cell-free translation system in the presence of Cy5-puro, a fluorescent puromycin, using mRNA without a stop codon. The interaction of proteins thus prepared was visualized using a single-molecule imaging technique. As a demonstration of this method, a motor protein, kinesin, was labeled with Cy5-puro at an efficiency of about 90%, and the processive movement of kinesin along microtubules was observed by using total internal reflection microscopy. It took only 2 h from the synthesis of proteins to the functional analysis. This method is applicable to the functional analysis of various kinds of proteins.

【 授权许可】

Unknown