Cell-free expression of G-protein-coupled receptors.
Cell-free expression of G-protein-coupled receptors.
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DOI：
10.1007/978-1-4939-2230-7_10
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发表时间：
2015-01-01
期刊：
影响因子：
--
通讯作者：
Bernhard, Frank
中科院分区：
文献类型：
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作者：
Orban, Erika;Proverbio, Davide;Bernhard, Frank
无细胞表达已成为膜蛋白生产的新标准。无细胞系统中表达复杂性的降低消除了中心瓶颈，并允许可靠和有效地合成许多不同类型的膜蛋白。此外，无细胞反应的开放可及性使得无细胞表达的膜蛋白能够在各种各样的供应的添加剂中共翻译溶解。因此，可以根据单个膜蛋白靶标的要求来调节疏水环境。我们提出了不同的方法制备规模的无细胞生产的G-蛋白偶联受体使用大肠杆菌细胞提取物。我们研究了实施去污剂、纳米盘或脂质体的表达条件。产生的蛋白质样品可以直接用于进一步的功能表征。
Cell-free expression has emerged as a new standard for the production of membrane proteins. The reduction of expression complexity in cell-free systems eliminates central bottlenecks and allows the reliable and efficient synthesis of many different types of membrane proteins. Furthermore, the open accessibility of cell-free reactions enables the co-translational solubilization of cell-free expressed membrane proteins in a large variety of supplied additives. Hydrophobic environments can therefore be adjusted according to the requirements of individual membrane protein targets. We present different approaches for the preparative scale cell-free production of G-protein-coupled receptors using the extracts of Escherichia coli cells. We exemplify expression conditions implementing detergents, nanodiscs, or liposomes. The generated protein samples could be directly used for further functional characterization.