Abstract

Summary 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 (AT 1 R) on amino-functionalized polystyrene microspheres. We identified a selective DNA aptamer of AT 1 R by a column-based SELEX approach with micromolar affinity. Two single-stranded DNA strands were utilized to introduce the AT 1 R aptamer and angiotensin II 3-8 peptide to the microsphere surface, resulting in the two surface-positioned sites. The two-point immobilized AT 1 R 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 AT 1 R 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.