Construction of α1A-Adrenoceptor-Based Stationary Phase Triggered by the Ultrahigh Affinity Protein Pair Colicin E7 Dnase /IM7 for Targeted Bioactive Compound Screening in Herba leonuri.

Immobilization of functional proteins onto solid surfaces is pivotal to propel the advancement of biomaterials in both biomedical and industrial technologies. However, the process has been hindered when it comes to G protein-coupled receptors due to the homogeneity of the seven transmembrane proteins. Herein, we exploited the efficient ultrahigh affinity of colicin E7 (CL7) and its cognate immunity protein IM7 for conjugation of functional protein onto macro-porous silica gel and established a robust chromatographic system for separation of compounds from complex matrices. Benefiting from the ultrahigh affinity nature of the protein pair, conjugation of α1A-adrenoceptor (α1A-AR) was achieved at ambient conditions in less than 20 min with high specificity and stability. Feasibility of the platform was tested by determining the binding parameters between drugs and receptor under various conditions (different pHs and concentration of methanol). Under the desired condition, carvedilol gave the association constant of (11.56±0.7) × 105 M-1 indicating immobilization of α1A-AR via this method still enhances its binding activity to drugs. Furthermore, stachydrine and leonurine were obtained as the bioactive compounds targeting the immobilized α1A-AR. Our study is expected to offer a versatile platform to achieve effective and specific immobilization of other functional proteins, thus enabling easy separation of the target compound from complex systems.