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首页> 外文期刊>The FEBS journal >Direct integration of cell-free-synthesized connexin-43 into liposomes and hemichannel formation
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Direct integration of cell-free-synthesized connexin-43 into liposomes and hemichannel formation

机译：无细胞合成的连接蛋白43直接整合到脂质体和半通道形成中

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摘要

Proteoliposomes were directly prepared by synthesizing membrane proteins with the use of minimal protein synthesis factors isolated from Escherichia coli (the PURE system) in the presence of liposomes. Connexin-43 (Cx43), which is a water-insoluble integral membrane protein that forms a hexameric complex in membranes, was cotranslationally integrated with an essentially uniform orientation in liposomes. The addition of liposomes following protein expression (post-translational presence of liposomes) did not lead to the integration of Cx43 into the liposome membranes. The amount of integrated Cx43 increased as the liposome concentration increased. The presence of liposomes did not influence the total amount of synthesized Cx43. The Cx43 integrated into the liposome membranes formed open membrane pores. These results indicate that the liposomes act in a chaperone-like manner by preventing Cx43 from aggregating in solution, because of integration into the bilayer, and also by functionalization of the integrated Cx43 in the membrane. This is the first report that cell-free- synthesized water-insoluble membrane protein is directly integrated with a uniform orientation as a functional oligomer into liposome membranes. This simple proteoliposome preparation procedure should be a valuable approach for structural and functional studies of membrane proteins.
机译：通过使用在脂质体存在下从大肠杆菌（PURE系统）中分离的最小蛋白质合成因子合成膜蛋白，直接制备蛋白脂质体。连接蛋白-43（Cx43）是一种不溶于水的整体膜蛋白，可在膜中形成六聚体复合物，并在脂质体中以基本一致的方向共翻译整合。在蛋白质表达后添加脂质体（翻译后存在脂质体）不会导致Cx43整合到脂质体膜中。随着脂质体浓度的增加，整合的Cx43的量增加。脂质体的存在不影响合成的Cx43的总量。整合到脂质体膜中的Cx43形成了开放的膜孔。这些结果表明脂质体以分子伴侣的方式发挥作用，原因是由于其结合到双层中，防止了Cx43在溶液中的聚集，而且还通过整合了膜中Cx43的功能化。这是第一个报道，无细胞合成的水不溶性膜蛋白以功能性寡聚物的均匀取向直接整合到脂质体膜中。这种简单的蛋白脂质体制备程序应该是用于膜蛋白结构和功能研究的有价值的方法。

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