Cell-free synthesis of a functional ion channel in the absence of a membrane and in the presence of detergent
Cell-free synthesis of a functional ion channel in the absence of a membrane and in the presence of detergent
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DOI：
10.1021/bi049049y
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发表时间：
2004-10-05
期刊：
影响因子：
2.9
通讯作者：
Ghazi, A
中科院分区：
文献类型：
--
作者：
Berrier, C;Park, KH;Ghazi, A
我们已经研究了在没有膜和存在洗涤剂的情况下，细胞膜蛋白合成的可能性。我们使用细菌的机械敏感通道MscL，一个homopentamer，作为模型蛋白。广泛的非离子或两性离子洗涤剂，Triton X-100，吐温20，Brij 58 p，正十二烷基β-D-麦芽糖苷，和CHAPS，与无细胞合成兼容，而正辛基β-D-葡萄糖苷和脱氧胆酸盐具有抑制作用。在Triton X-100存在下的体外合成产生毫克量的MscL/毫升裂解物。交联实验表明，蛋白质能够寡聚在洗涤剂。当纯化的蛋白质在脂质体中重组并通过膜片钳技术研究时，其在单分子水平上的活性与大肠杆菌中产生的重组蛋白质的活性相似。膜蛋白的无细胞合成应该证明是一种有价值的工具，用于生产在异源系统中过表达是困难的膜蛋白。
We have investigated the possibility of cell-fee synthesis of membrane proteins in the absence of a membrane and in the presence of detergent. We used the bacterial mechanosensitive channel MscL, a homopentamer, as a model protein. A wide range of nonionic or zwitterionic detergents, Triton X-100, Tween 20, Brij 58p, n-dodecyl beta-D-maltoside, and CHAPS, were compatible with cell-free synthesis, while n-octyl beta-D-glucoside and deoxycholate had an inhibitory effect. In vitro synthesis in the presence of Triton X-100 yielded milligram amounts of MscL per milliliter of lysate. Cross-linking experiments showed that the protein was able to oligomerize in detergents. When the purified protein was reconstituted in liposomes and studied by the patch-clamp technique, its activity at the single-molecule level was similar to that of the recombinant protein produced in Escherichia coli. Cell-free synthesis of membrane proteins should prove a valuable tool for the production of membrane proteins whose overexpression in heterologous systems is difficult.