Highly efficient GPCR immobilization with enhanced fouling resistance, salt tolerance, and chromatographic performance

Colloids Surf B Biointerfaces. 2024 Feb 22;236:113818. doi: 10.1016/j.colsurfb.2024.113818. Online ahead of print.ABSTRACTThe feasibility of immobilized protein-based biodetection relies critically on the activity of the immobilized proteins as well as the biocompatibility of the protein surface. Although many protein immobilization strategies have been developed with satisfied detection readout signals. Non-specific interactions caused by the protein-coating surface are still of great concern since they often interfere with or affect the reliability of detection. Herein, we developed a highly efficient G protein-coupled receptor (GPCR) immobilization method by the combination of polyethylene glycol (PEG) with a self-labeling enzyme-catalyzed reaction. The immobilization relies on the covalent interaction between the fusion tag of a target GPCR (kinase domain of epidermal growth factor receptor, EGFR) and its covalent inhibitor ibrutinib, which is modified on PEGylated silica gels. Two types of GPCRs, N-methyl-D-aspartate 2 A receptor (NMDAR2A) and endothelin A receptor (ETAR), were used as examples to realize protein immobilization. The GPCR modified gels and the affinity columns packed with them have been extensively characterized, in terms of non-specific adsorptions, retention factor (k'), half peak width (W1/2), tailing factor (Tf), theoretical plates (N), and association and dissociation constants of the ligands with the receptors. The immobilized GPCRs with reduced non...
Source: Colloids and Surfaces - Category: Biotechnology Authors: Source Type: research