Abstract: According to the codon preference of Escherichia coli, the β2 adrenergic receptor (β2AR) gene sequence was designed and synthesized. Then the E. coli-based cell-free protein synthesis (CFPS) system and MagneHis? Ni-Particles were used for efficient expression and purification. The purified receptor was identified by SDS-PAGE analysis and enzyme-linked receptor assays (ELRA). The results indicated that the codon adaptation index (CAI) of the optimized β2AR gene was 0.96 and its GC content was decreased from 58% to 46.17%, favoring its expression in E. coli. The optimal Mg2+ concentration in the expression system was 22 mmol/L, leading to the maximum protein expression of 1 250 μg/mL. SDS-PAGE revealed the specific band of the purified protein of 47 kDa with a purity of over 90% as expected. The results of direct ELRA showed that when the plates were coated with the receptor at 1:500 dilution, the OD values of the purified receptor binding to three horse radish peroxidase (HRP)-β-agonists decreased in the following order: clenbuterol, salbutamol, and ractopamine, which were 0.976, 0.836 and 0.728, respectively. β2AR was successfully synthesized by using the CFPS system, which will provide a theoretical and practical foundation for the rapid multi-residue determination of β-agonists based on the receptor.

Key words: β2 adrenergic receptor, codon optimization, E. coli-based cell-free protein synthesis system, expression, detection of β-agonists