In Vitro Reconstitution Platforms of Mammalian Cell-Free Expressed Membrane Proteins.
In Vitro Reconstitution Platforms of Mammalian Cell-Free Expressed Membrane Proteins.
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DOI：
10.1007/978-1-0716-1998-8_6
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Liu AP
中科院分区：
文献类型：
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作者：
Moghimianavval H;Hsu YY;Groaz A;Liu AP
膜蛋白是细胞膜中的重要组成部分，使细胞能够与其外部环境进行交流并进行细胞内信号传导。使用无细胞表达（CFE）系统的复杂膜蛋白的功能重建已被证明是具有挑战性的，主要是由于缺乏用于新生膜蛋白的正确折叠和易位以及它们向所提供的合成双层的递送的必要机制。在这里，我们提供了使用HeLa为基础的CFE系统和大纲分析研究其膜插入，拓扑结构，和他们的方向纳入支持的脂质双层或双层的巨型单层囊泡以及方法分离功能易位无细胞产生的膜蛋白的无细胞重建的功能性膜蛋白的协议。
Membrane proteins are essential components in cell membranes and enable cells to communicate with their outside environment and to carry out intracellular signaling. Functional reconstitution of complex membrane proteins using cell-free expression (CFE) systems has been proved to be challenging mainly due to the lack of necessary machinery for proper folding and translocation of nascent membrane proteins and their delivery to the supplied synthetic bilayers. Here, we provide protocols for detergent-free cell-free reconstitution of functional membrane proteins using HeLa-based CFE system and outline assays for studying their membrane insertion, topology, and their orientation upon incorporation into the supported lipid bilayers or bilayers of giant unilamellar vesicles as well as methods to isolate functional translocated cell-free produced membrane proteins.