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Site-Specific Immobilization of β₂-AR Using O⁶-Benzylguanine Derivative-Functionalized Supporter for High-Throughput Receptor-Targeting Lead Discovery

Wang, Jing, Wang, Yuxin, Liu, Jiajun, Li, Qian, Yin, Guowei, Zhang, Yajun, Xiao, Chaoni, Fan, Taiping, Zhao, Xinfeng, Zheng, Xiaohui
Analytical chemistry 2019 v.91 no.11 pp. 7385-7393
DNA repair, Escherichia coli, G-protein coupled receptors, bioactive compounds, chromatography, drugs, humans, ligands, mass spectrometry, polyacrylamide, polyethylene glycol, recipes, rosmarinic acid, screening, traditional medicine, transferases
The past decade has witnessed the great promise of strategies for ligand discovery based on surface-immobilized GPCRs. We present here a method for preparation of immobilized GPCRs. Key features include covalent immobilization with high specificity and robust application in drug-receptor interaction analysis and ligand screening. In our example assay using beta₂-adrenergic receptor (β₂-AR), the human DNA repair protein O⁶-alkylguanine-DNA alkyltransferase (hAGT) fusion receptor expressed in Escherichia coli was directly captured onto polyethylene glycol polyacrylamide (PEGA) resin. We observed even distribution and physiological functions of β₂-AR on the resin. The immobilized β₂-AR as a stationary phase enabled us to rapidly determine the binding of four drugs to β₂-AR. By coupling this assay to mass spectrometry, we screened rosmarinic acid as a bioactive compound targeting β₂-AR in Fructus Perillae. We concluded that O⁶-benzylguanine derivative-functionalized supporter is promising for specific immobilization of hAGT-tagged proteins; immobilized receptor chromatography has great potential in screening receptor-binding leads from herbal plants or traditional medicine recipes.