Anabuki Tomoaki, Tsukahara Miu, Okamoto Masanori, Matsuura Hideyuki, Takahashi Kosaku
Research Faculty of Agriculture, Hokkaido University, Kita9 Nishi9, Kita-ku, Sapporo 060-8589, Japan.
Center for Bioscience Research and Education, Utsunomiya University, 350 Mine-cho, Utsunomiya 321-8505, Japan.
Bioorg Med Chem Lett. 2018 Feb 15;28(4):783-786. doi: 10.1016/j.bmcl.2017.12.055. Epub 2017 Dec 26.
We synthesized a novel linker (1) with biotin, alkyne and amino groups for the identification of target proteins using a small molecule that contains an azide group (azide probe). The alkyne in the linker bound the azide probe via an azide-alkyne Huisgen cycloaddition. A protein cross-linker effectively bound the conjugate of the linker and an azide probe with a target protein. The covalently bound complex was detected by western blotting. Linker 1 was applied to a model system using an abscisic acid receptor, RCAR/PYR/PYL (PYL). Cross-linked complexes of linker 1, the azide probes and the target proteins were successfully visualized by western blotting. This method of target protein identification was more effective than a previously developed method that uses a second linker with biotin, alkyne, and benzophenone (linker 2) that acts to photo-crosslink target proteins. The system developed in this study is a method for identifying the target proteins of small bioactive molecules and is different from photo-affinity labelling.
我们合成了一种带有生物素、炔基和氨基的新型连接子（1），用于使用含有叠氮基团的小分子（叠氮探针）鉴定靶蛋白。连接子中的炔基通过叠氮-炔基胡伊斯根环加成反应与叠氮探针结合。一种蛋白质交联剂有效地将连接子与叠氮探针的共轭物与靶蛋白结合。通过蛋白质免疫印迹法检测共价结合的复合物。连接子1应用于使用脱落酸受体RCAR/PYR/PYL（PYL）的模型系统。通过蛋白质免疫印迹法成功可视化了连接子1、叠氮探针和靶蛋白的交联复合物。这种鉴定靶蛋白的方法比先前开发的使用带有生物素、炔基和二苯甲酮的第二种连接子（连接子2）对靶蛋白进行光交联的方法更有效。本研究中开发的系统是一种鉴定小生物活性分子靶蛋白的方法，与光亲和标记不同。
