Enhancing Chromatographic Performance of Immobilized Angiotensin II Type 1 Receptor by Strain-Promoted Alkyne Azide Cycloaddition through Genetically Encoded Unnatural Amino Acid.

Abstract

During integration to the solid surface, the effects of tags introduced for bioorthogonal reactions on protein activity have received far less investigation. This represents the major challenge of improving the performance of the immobilized protein-based assays. Herein, the relationship between the fusion tags and their reaction efficiency in mediating the assay performance was realized by determining the chromatographic performance using genetically encoded azide-alkyne cycloaddition, and Halo- and SNAP-tagged bioorthogonal reactions for synthesizing immobilized angiotensin II type 1 receptor (AT1R). We demonstrated that immobilization with the incorporation of unnatural amino acid in the receptor minimizes the peak tailings and broadenings of irbesartan, fimasartan, losartan, and tasosartan, while attachment via large tags (SNAP and Halo) leads to serious asymmetry peaks. Upon the first immobilization, the association constants of the four drugs to AT1R appeared to be 1 order of magnitude greater than the other two attachments. Such enhancement is likely reasoned by the improved association rate constants and the relatively identical dissociation rates due to the small tag. While demonstrating improved chromatographic performance, the immobilized AT1R prepared by the genetically encoded azide-alkyne reaction was applied in analyzing Uncaria Schreber nom. cons. extract, which identified hynchophylline as a specific ligand binding to the receptor. As immobilized proteins move toward diverse assays, our findings provide an unprecedented insight into the relation between fusion tags and their reaction efficiency in mediating the assay performance, which is thus dedicated to the creation of a protein-functionalized surface for precisely determining the drug-protein interaction and discovering the specific partner of the protein.