A universal method for surface-based binding assays by preparing immobilized β-adrenergic receptor stationary phase using solid binding peptide as a fusion tag.

Protein functionalized surface has the potential to develop new assays for determining the drug-like properties of potential compounds and discovering specific partners of G protein-coupled receptors (GPCRs). However, a universal method for purifying and immobilizing functional GPCRs has remained elusive. To this end, we developed a general and rapid way to purify and immobilize β-adrenergic receptor (βAR) by silicon-specific peptide. We screened CotB1p as a tag from six silica-binding peptides (minTBP-1, CotB1p, SB, Car, and Si) by examining their affinity to macroporous silica gel. We investigated the adsorption and desorption of CotB1p-tagged β-adrenoceptor (βAR-CotB1p) under diverse conditions to propose a protocol for receptor purification and immobilization. Under optimized conditions, βAR immobilization were achieved by directly immersing cell lysates harboring the receptor with silica gel, and the elution of the receptor without demonstratable contaminants was realized by including l-arginine/L-lysine in the elutes. This allows purification of the receptor from Escherichia coli (E.coli) lysates with a purity of 95 %. The immobilized receptor was utilized as a stationary phase to reveal the tag impact on ligand-binding outputs by comparing the CotB1p-strategy with a typical covalent method. The Ks of salbutamol, chlorprenaline, tulobuterol, and terbutaline on βAR-CotB1p column were 1.26 × 10, 6.59 × 10, 7.90 × 10, and 8.97 × 10 M respectively, which were two orders of magnitude higher than those on the Halo-βAR column. The whole immobilization was accomplished within 30 min without the requirement of any special treatment, resulting in enhanced accuracy for determining receptor-ligand binding parameters. Taken together, CotB1p-mediated strategy is simple, rapid, and universal for purification or immobilization of unstable biomolecules like GPCRs for analytical and biological applications.