A Tandem-Affinity Purification Method for Identification of Primary Intracellular Drug-Binding Proteins.

ABSTRACT: 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 spe

SUBMITTER: Islam S 

PROVIDER: S-EPMC10878392 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature