{'fraction_non_alphanumeric': 0.038852361028093245, 'fraction_numerical': 0.021518230723251645, 'mean_word_length': 5.590551181102362, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
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 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.