Preparation of the immobilized α1A-adrenoceptor column by the ultra-high affinity protein pair CL7/Im7 and its application in drug-protein interaction analysis

Abstract

Immobilizing the target protein on a solid surface with controlled orientation, high specificity, and maintained activity is a proven strategy to enhance the stability of the protein. In this study, we employed an ultra-high affinity protein pair consisting of a mutant of colicin E7 Dnase and its corresponding inhibitor, immunity protein 7(Im7), to develop an immobilized α_1A-adrenoceptor (α_1A-AR) column. Briefly, we expressed α_1A-AR fused with CL7 as a tag at its C-terminus in Escherichia coli cells. Meanwhile, we got His-tagged Im7 at the same manner. Following purification, the His-tagged Im7 was utilized to functionalize the macro-porous silica gel. Leveraging the ultra-high affinity between the protein pair, we achieved robust and specific covalent immobilization of α_1A-AR covalently at ambient conditions in buffer solutions, without the requirement for additional regents. The successful immobilization of the receptor, without extraneous protein adsorption, was confirmed through X ray photoelectron spectroscopy and chromatographic investigations. Frontal analysis and adsorption energy distribution analysis further validated the feasibility of the immobilization method. Our findings align well with those reported in the literature. This work is poised to provide a modular platform for conducting effective investigations into the binding interactions between other functional proteins and the drugs.

Keywords: Adsorption energy distribution; Binding interactions; Frontal analysis; Immobilized receptor chromatography; Ultra-high affinity protein pair: CL7/Im7; α(1A)-adrenoceptor.