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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.