A universal method for surface-based binding assays by preparing immobilized β2-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 2 -adrenergic receptor (β 2 AR) by silicon-specific peptide. We screened CotB1p as a tag from six silica-binding peptides (minTBP-1, CotB1p, SB 7 , Car 9 , and Si 4–1 ) by examining their affinity to macroporous silica gel. We investigated the adsorption and desorption of CotB1p-tagged β 2 -adrenoceptor (β 2 AR-CotB1p) under diverse conditions to propose a protocol for receptor purification and immobilization. Under optimized conditions, β 2 AR 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 K A s of salbutamol, chlorprenaline, tulobuterol, and terbutaline on β 2 AR-CotB1p column were 1.26 × 10 6 , 6.59 × 10 6 , 7.90 × 10 6 , and 8.97 × 10 5 M −1 respectively, which were two orders of magnitude higher than those on the Halo-β 2 AR 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.
危险品化学品经营许可证（带存储）