Identification of recombinant AtPYL2, an abscisic acid receptor, in E. coli using a substrate-derived bioactive small molecule, a biotin linker with alkyne and amino groups, and a protein cross-linker
Identification of recombinant AtPYL2, an abscisic acid receptor, in E. coli using a substrate-derived bioactive small molecule, a biotin linker with alkyne and amino groups, and a protein cross-linker
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DOI：
10.1016/j.bmcl.2019.126634
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发表时间：
2019-11-01
影响因子：
2.7
通讯作者：
Takahashi, Kosaku
中科院分区：
文献类型：
--
作者：
Anabuki, Tomoaki;Ito, Yusuke;Takahashi, Kosaku
生物活性小分子靶蛋白的鉴定是正向化学遗传学的重要研究内容之一。使用与小分子结合的树脂的亲和层析技术经常用于鉴定生物活性小分子的靶蛋白。在这里，我们报告了一种新的方法来分离与生物活性小分子靶向的蛋白质，使用具有炔和氨基基团的生物素接头，含有二硫键的蛋白质交联剂，和具有叠氮基的生物活性小分子（叠氮基探针）。叠氮探针与靶蛋白结合后，靶蛋白与叠氮探针的复合物通过生物素接头通过叠氮-炔Huisgen环加成和含二硫键的蛋白交联剂共价结合。将该三元复合物固定在具有链霉亲和素的亲和基质上，然后用含有用于切割二硫键的还原剂的缓冲液选择性洗脱靶蛋白。该方法使用具有叠氮基的探针，该叠氮基是小的官能团，并且有可能成为克服引入到探针中的官能团的阻碍的解决方案策略，该阻碍减少了与靶蛋白的结合。使用接头1、3 '-叠氮基脱落酸3和含有二硫键的蛋白质交联剂（DTSSP 5）显示了该方法在本研究中的有效性。
Target protein identification of bioactive small molecules is one of the most important research in forward chemical genetics. The affinity chromatography technique to use a resin bound with a small molecule is often used for identification of a target protein of a bioactive small molecule. Here we report a new method to isolate a protein targeted with a bioactive small molecule using a biotin linker with alkyne and amino groups, protein cross-linker containing disulfide bond, and a bioactive small molecule with an azido group (azido probe). After an azido probe is associated with a target protein, the complex of a target protein and azido probe is covalently bound through the biotin linker by azide-alkyne Huisgen cycloaddition and protein cross-linker containing disulfide bond. This ternary complex is immobilized on an affinity matrix with streptavidin, and then the target protein is selectively eluted with a buffer containing a reducing agent for cleavage of disulfide bonds. This method uses a probe having an azido group, which a small functional group, and has the possibility to be a solution strategy to overcome the hindrance of a functional group introduced into the probe that reduces association a target protein. The effectiveness of the method in this study was shown using linker 1, 3'-azidoabscisic acid 3, and protein cross-linker containing a disulfide bond (DTSSP 5).