Abstract
Methods of immobilized proteins are challenged by the way how to capture the proteins in their intact functional states. Here we present a two-point, high-specific method for the immobilization of conformationally specific angiotensin II type 1 receptor (AT1R) on amino-functionalized polystyrene microspheres. We identified a selective DNA aptamer of AT1R by a column-based SELEX approach with micromolar affinity. Two single-stranded DNA strands were utilized to introduce the AT1R aptamer and angiotensin II 3-8 peptide to the microsphere surface, resulting in the two surface-positioned sites. The two-point immobilized AT1R exhibited enhanced ligand-binding activity and stability in comparison with that prepared by a one-positioned site. Ginsenoside Rg1 and rosmarinic acid were screened from the herbal extract and proved to bind with AT1R through the allosteric and orthosteric sites of the receptor, respectively. These provide a generally applicable approach for functional protein immobilization with enhanced conformation stability, ligand binding activity, and screening efficiency.