The synthesis and high-level expression of a β2-adrenergic receptor gene in a tetracycline-inducible stable mammalian cell line

High-level expression of G-protein-coupled receptors (GPCRs) in functional form is required for structure-function studies. The main goal of the present work was to improve expression levels of beta(2)-adrenergic receptor (beta(2)-AR) so that biophysical studies involving EPR, NMR, and crystallography can be pursued. Toward this objective, the total synthesis of a codon-optimized hamster beta(2)-AR gene suitable for high-level expression in mammalian systems has been accomplished. Transient expression of the gene in COS-1 cells resulted in 18 +/- 3 pmol beta(2)-AR/mg of membrane protein, as measured by saturation binding assay using the beta(2)-AR antagonist [ 3 H] dihydroalprenolol. Previously, we reported the development of an HEK293S tetracycline-inducible system for high-level expression of rhodopsin. Here, we describe construction of beta(2)-AR stable cell lines using the HEK293S-TetR-inducible system, which, after induction, express wild-type beta(2)-AR at levels of 220 +/- 40 pmol/mg of membrane protein corresponding to 50 +/- 8 mg/15-cm plate. This level of expression is the highest reported so far for any wild-type GPCR, other than rhodopsin. The yield of functional receptor using the single-step affinity purification is 12 +/- 3 mg/15-cm plate. This level of expression now makes it feasible to pursue structure-function studies using EPR. Furthermore, scale-up of beta(2)-AR expression using suspension cultures in a bioreactor should now allow production of enough beta(2)-AR for the application of biophysical techniques such as NMR spectroscopy and crystallography.