串联亲和纯化
生物素化
计算生物学
药物发现
免疫沉淀
药品
血浆蛋白结合
靶蛋白
化学
链霉亲和素
生物化学
亲和层析
生物
生物素
药理学
酶
基因
作者
Sehbanul Islam,Jitendra Gour,T. M. Beer,Hsin‐Yao Tang,Joel Cassel,Joseph M. Salvino,Luca Busino
标识
DOI:10.1021/acschembio.3c00570
摘要
In the field of drug discovery, understanding how small molecule drugs interact with cellular components is crucial. Our study introduces a novel methodology to uncover primary drug targets using Tandem Affinity Purification for identification of Drug-Binding Proteins (TAP-DBP). Central to our approach is the generation of a FLAG-hemagglutinin (HA)-tagged chimeric protein featuring the FKBP12(F36V) adaptor protein and the TurboID enzyme. Conjugation of drug molecules with the FKBP12(F36V) ligand allows for the coordinated recruitment of drug-binding partners effectively enabling in-cell TurboID-mediated biotinylation. By employing a tandem affinity purification protocol based on FLAG-immunoprecipitation and streptavidin pulldown, alongside mass spectrometry analysis, TAP-DBP allows for the precise identification of drug-primary binding partners. Overall, this study introduces a systematic, unbiased method for identification of drug-protein interactions, contributing a clear understanding of target engagement and drug selectivity to advance the mode of action of a drug in cells.
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