The synthesis and high‐level expression of a β2‐adrenergic receptor gene in a tetracycline‐inducible stable mammalian cell line
Open Access
- 1 June 2006
- journal article
- Published by Wiley in Protein Science
- Vol. 15 (6), 1433-1440
- https://doi.org/10.1110/ps.062080006
Abstract
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 β2-adrenergic receptor (β2-AR) so that biophysical studies involving EPR, NMR, and crystallography can be pursued. Toward this objective, the total synthesis of a codon-optimized hamster β2-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 β2-AR/mg of membrane protein, as measured by saturation binding assay using the β2-AR antagonist [3H] dihydroalprenolol. Previously, we reported the development of an HEK293S tetracycline-inducible system for high-level expression of rhodopsin. Here, we describe construction of β2-AR stable cell lines using the HEK293S-TetR-inducible system, which, after induction, express wild-type β2-AR at levels of 220 ± 40 pmol/mg of membrane protein corresponding to 50 ± 8 μg/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 μg/15-cm plate. This level of expression now makes it feasible to pursue structure–function studies using EPR. Furthermore, scale-up of β2-AR expression using suspension cultures in a bioreactor should now allow production of enough β2-AR for the application of biophysical techniques such as NMR spectroscopy and crystallography.Keywords
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