CEA, DSV, iRTSV, Biopuces, Grenoble, France.
J Membr Biol. 2010 Feb;233(1-3):85-92. doi: 10.1007/s00232-010-9227-8. Epub 2010 Feb 5.
A simple method for the reconstitution of membrane protein from submicron proteoliposomes into giant unilamellar vesicles (GUVs) is presented here: This method does not require detergents, fusion peptides or a dehydration step of the membrane protein solution. In a first step, GUVs of lipids were formed by electroformation, purified and concentrated; and in a second step, the concentrated GUV solution was added to a small volume of vesicles or proteoliposomes. Material transfer from submicron vesicles and proteoliposomes to GUVs occurred spontaneously and was characterized with fluorescent microscopy and patch-clamp recordings. As a functional test, the voltage-dependent, anion-selective channel protein was reconstituted into GUVs, and its electrophysiological activity was monitored with the patch clamp. This method is versatile since it is independent of the presence of the protein, as demonstrated by the fusion of fluorescently labeled submicron vesicles and proteoliposomes with GUVs.
这里介绍了一种将膜蛋白从小型脂双层囊泡（submicron proteoliposomes）复性为大单室囊泡（giant unilamellar vesicles，GUVs）的简单方法：该方法不需要去污剂、融合肽或膜蛋白溶液的脱水步骤。在第一步中，通过电形成制备 GUVs，并对其进行纯化和浓缩；在第二步中，将浓缩的 GUV 溶液加入到少量的囊泡或脂双层囊泡中。从小型囊泡和脂双层囊泡向 GUV 的物质转移是自发发生的，并通过荧光显微镜和膜片钳记录进行了表征。作为功能测试，将电压依赖性阴离子选择性通道蛋白复性到 GUVs 中，并通过膜片钳监测其电生理活性。该方法具有通用性，因为它独立于蛋白质的存在，这可以通过与 GUVs 的融合来证明，融合的是荧光标记的小型脂双层囊泡和脂双层囊泡。
