Abstract: Codon optimization, construction of recombinant baculovirus system (Bac-to-Bac), screening of optimal expression conditions and Ni-chelating affinity chromatography were used to develop an efficient strategy for the expression and purification of β2 adrenergic receptor（β2AR）gene in Sf9 cells. The modified β2AR gene was artificially synthesized and cloned to the transfer vector pFastBac1 to construct the recombinant baculovirus expression plasmid pFastBac1-β2AR′. Then Sf9 cells were transfected with the recombinant plasmid. The expression conditions were optimized. The expression product was purified by Ni-NTA-affinity chromatography and its ligand binding affinity was determined by enzyme-linked immunosorbent assay (ELISA). The results showed that the optimal expression conditions were as follows: multiplicity of infection (MOI) of the infected cells, 5; and expression time, 48 h. In the Western blot analysis, a single band with an apparent molecular mass of 47 kD appeared as expected. After purification, the recombinant protein was more than 90% pure. The ligand binding assay indicated that it could specifically bind to all three horseradish peroxidase (HRP)-β-agonists: clenbuterol, salbutamol, ractopamine, and the OD values obtained from ELISA were 0.983, 0.947 and 0.912, respectively. In this paper, the efficient expression of β2AR in Sf9 cells was accomplished. Furthermore, the purified receptor protein remained better binding affinity to β-agonists, laying a foundation for developing a rapid multi-residue assay for the determination of β-agonists with β2AR.

Key words: β2 adrenergic receptor, baculovirus expression vector, Sf9 cells, expression, purification