The synthesis and high-level expression of a β2-adrenergic receptor gene in a tetracycline-inducible stable mammalian cell line
The synthesis and high-level expression of a β2-adrenergic receptor gene in a tetracycline-inducible stable mammalian cell line
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DOI：
10.1110/ps.062080006
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发表时间：
2006-06-01
期刊：
影响因子：
8
通讯作者：
Khorana, H. Gobind
中科院分区：
文献类型：
--
作者：
Chelikani, Prashen;Reeves, Philip J.;Khorana, H. Gobind
结构功能研究需要以功能形式高水平表达 G 蛋白偶联受体 (GPCR)。目前工作的主要目标是提高β(2)-肾上腺素能受体(β(2)-AR)的表达水平，以便可以进行涉及EPR、NMR和晶体学的生物物理研究。为了实现这一目标，适合在哺乳动物系统中高水平表达的密码子优化的仓鼠β(2)-AR基因的全合成已经完成。该基因在 COS-1 细胞中的瞬时表达导致 18 +/- 3 pmol β(2)-AR/mg 膜蛋白，使用 β(2)-AR 拮抗剂 [ 3 H]二氢阿普洛尔通过饱和结合测定进行测量。之前，我们报道了用于高水平表达视紫红质的 HEK293S 四环素诱导系统的开发。在这里，我们描述了使用 HEK293S-TetR 诱导系统构建 β(2)-AR 稳定细胞系，该系统在诱导后以 220 +/- 40 pmol/mg 膜蛋白水平表达野生型 β(2)-AR，对应于 50 +/- 8 mg/15-cm 板。这一表达水平是迄今为止除视紫红质外所有野生型 GPCR 所报道的最高表达水平。使用单步亲和纯化的功能性受体的产量为 12 +/- 3 mg/15-cm 板。这种表达水平现在使得使用 EPR 进行结构功能研究成为可能。此外，在生物反应器中使用悬浮培养物放大β(2)-AR表达现在应该能够产生足够的β(2)-AR用于生物物理技术的应用，例如核磁共振波谱学和晶体学。
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.