Reversible and site-specific immobilization of β-adrenergic receptor by aptamer-directed method for receptor-drug interaction analysis.

Immobilized protein makes a profound impact on the development of assays for drug discovery, diagnosis and in vivo biological interaction analysis. Traditional methods are enormously challenged by the G-protein coupled receptor ascribed to the loss of receptor functions. We introduced a β-adrenergic receptor (β-AR) aptamer into the immobilization of the receptor. This was achieved by mixing the receptor conjugated silica gel with cell lysates containing the receptor. We found that the aptamer-directed method makes immobilized β-AR good stability in seven days and high specificity of ligand recognition at the subtype receptor level. Feasibility of the immobilized β-AR in drug-receptor interaction analysis was evaluated by injection amount-dependent method, nonlinear chromatography, and peak decay analysis. Salbutamol, methoxyphenamine, ephedrine hydrochloride, clorprenaline, tulobuterol, bambuterol, propranolol and ICI 118551 bound to the receptor through one type of binding sites. The association constants presented good agreement within the three methods but exhibited clear differences from the data by radio-ligand binding assay. Regarding these results, we concluded that the aptamer-directed method will probably become an alternative for reversible and site-specific immobilization of GPCRs directly from complex matrices; the immobilized receptor is qualitative for drug-receptor interaction analysis.