In vitro membrane protein synthesis inside Sec translocon-reconstituted cell-sized liposomes.
In vitro membrane protein synthesis inside Sec translocon-reconstituted cell-sized liposomes.
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DOI：
10.1038/srep36466
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发表时间：
2016-11-03
影响因子：
4.6
通讯作者：
Matsuura T
中科院分区：
文献类型：
--
作者：
Ohta N;Kato Y;Watanabe H;Mori H;Matsuura T
使用细胞大小的脂质体内的体外转录-翻译系统（IVTT）的蛋白质合成已经成为在细胞模拟条件下研究生物系统的性质的有价值的工具。然而，以前的脂质体系统缺乏膜蛋白易位的机制。在这里，我们重建了由来自大肠杆菌的SecYEG组成的易位子，在细胞大小的脂质体内。细胞大小的脂质体还携带重构的IVTT，从而为膜蛋白合成提供细胞模拟环境。利用E.大肠杆菌作为模型膜蛋白，我们发现，无论是在脂质体内合成的EmrE的量和活性增加约三倍，通过纳入Sec translocon。还鉴定了该易位子诱导的EmrE的拓扑结构变化。9株E.所测试的大肠杆菌内膜蛋白的表达量通过转座子的掺入而增加。通过引入Sec易位子，目的膜蛋白的膜整合效率增加，并且能够整合否则不能插入的膜蛋白。此外，这项工作代表了通过自下而上的方法构建人工细胞的重要一步。
Protein synthesis using an in vitro transcription-translation system (IVTT) inside cell-sized liposomes has become a valuable tool to study the properties of biological systems under cell-mimicking conditions. However, previous liposome systems lacked the machinery for membrane protein translocation. Here, we reconstituted the translocon consisting of SecYEG from Escherichia coli inside cell-sized liposomes. The cell-sized liposomes also carry the reconstituted IVTT, thereby providing a cell-mimicking environment for membrane protein synthesis. By using EmrE, a multidrug transporter from E. coli, as a model membrane protein, we found that both the amount and activity of EmrE synthesized inside the liposome is increased approximately three-fold by incorporating the Sec translocon. The topological change of EmrE induced by the translocon was also identified. The membrane integration of 6 out of 9 E. coli inner membrane proteins that was tested was increased by incorporation of the translocon. By introducing the Sec translocon, the membrane integration efficiency of the membrane protein of interest was increased, and enabled the integration of membrane proteins that otherwise cannot be inserted. In addition, this work represents an essential step toward the construction of an artificial cell through a bottom-up approach.
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影响因子：
9.9
作者：
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DOI：
10.1073/pnas.081617398
发表时间：
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影响因子：
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