Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4
Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4
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DOI：
10.1073/pnas.0811089106
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发表时间：
2009-07-21
影响因子：
11.1
通讯作者：
Zhang, Shuguang
中科院分区：
文献类型：
--
作者：
Cook, Brian L.;Steuerwald, Dirk;Zhang, Shuguang
尽管对嗅觉系统的理解在下游受体信号传导和嗅觉神经元的连接水平上取得了进展，但在受体及其与气味配体的相互作用和识别的分子水平上，对嗅觉系统的理解仍然很少。这些受体的结构和功能机制仍然是一个诱人的谜，因为许多以前的大规模生产功能性嗅觉受体（ORs）的尝试尚未成功。为了研究嗅觉的生物化学和分子机制，我们开发了一种哺乳动物表达系统，用于大规模生产和纯化一种毫克量的功能性OR蛋白。在这里，我们报道了从HEK293S四环素诱导系统中纯化的人OR17-4 （hOR17-4）的研究。通过生物反应器悬浮培养，实现了产量的放大，经免疫亲和层析和大小排斥层析，可以制备出bbb10 mg的hOR17-4单体受体，表达量达到3 mg/L培养基。利用质谱鉴定了几个关键的翻译后修饰，CD谱显示受体的α -螺旋结构接近50%，与最近确定的其他G蛋白偶联受体结构相似。通过表面等离子体共振测量，洗涤剂溶解的hOR17-4特异性结合其已知的激活气味剂百合和floralozone。hOR17-4还通过钙离子动员来识别异源细胞中的特定气味。我们的系统是可行的生产大量的或必要的结构和功能分析和研究或生物传感器设备。
Although understanding of the olfactory system has progressed at the level of downstream receptor signaling and the wiring of olfactory neurons, the system remains poorly understood at the molecular level of the receptors and their interaction with and recognition of odorant ligands. The structure and functional mechanisms of these receptors still remain a tantalizing enigma, because numerous previous attempts at the large-scale production of functional olfactory receptors (ORs) have not been successful to date. To investigate the elusive biochemistry and molecular mechanisms of olfaction, we have developed a mammalian expression system for the large-scale production and purification of a functional OR protein in milligram quantities. Here, we report the study of human OR17-4 (hOR17-4) purified from a HEK293S tetracycline-inducible system. Scale-up of production yield was achieved through suspension culture in a bioreactor, which enabled the preparation of > 10 mg of monomeric hOR17-4 receptor after immunoaffinity and size exclusion chromatography, with expression yields reaching 3 mg/L of culture medium. Several key post-translational modifications were identified using MS, and CD spectroscopy showed the receptor to be approximate to 50% alpha-helix, similar to other recently determined G protein-coupled receptor structures. Detergent-solubilized hOR17-4 specifically bound its known activating odorants lilial and floralozone in vitro, as measured by surface plasmon resonance. The hOR17-4 also recognized specific odorants in heterologous cells as determined by calcium ion mobilization. Our system is feasible for the production of large quantities of OR necessary for structural and functional analyses and research into OR biosensor devices.