A new automated technique for the reconstitution of hydrophobic proteins into planar bilayer membranes. Studies of human recombinant uncoupling protein 1
A new automated technique for the reconstitution of hydrophobic proteins into planar bilayer membranes. Studies of human recombinant uncoupling protein 1
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DOI：
10.1016/j.bbabio.2006.03.006
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发表时间：
2006-05-01
影响因子：
4.3
通讯作者：
Pohl, Elena E.
中科院分区：
文献类型：
--
作者：
Beck, Valeri;Jaburek, Martin;Pohl, Elena E.
大量的注释通道和运输蛋白在后基因组时代的电生理特性将大大促进了快速和强大的方法引入膜蛋白的功能纳入定义的脂质双层。在这里，我们描述了一种自动化的技术，用于重组成脂质双层膜的膜蛋白，这大大减少了重组时间和膜形成所需的蛋白质的量。该方法允许研究单个蛋白通道以及将多拷贝（类似于10（7））插入单个双层。尽管膜面积相对较大（直径可达300 μ m），但膜的高稳定性允许施加高达300 mV的跨膜电压。这一特征对于内膜线粒体蛋白的研究尤其重要，因为它们在生理条件下的作用电位高达200 mV。正是这些优点的结合，使得对质子转运蛋白典型的微小单个蛋白质电导的详细研究成为可能。我们应用新技术对在大肠杆菌中过表达的人重组解偶联蛋白1（hUCP 1）进行了重组和电生理学特性研究。大肠杆菌中表达，并从包涵体中纯化。我们证明，在脂肪酸的存在下，hUCP1的活性与从棕色脂肪组织中分离的UCP1的活性相当。(c)2006 Elsevier B.V.保留所有权利。
Electrophysiological characterisation of the vast number of annotated channel and transport proteins in the postgenomic era would be greatly facilitated by the introduction of rapid and robust methods for the functional incorporation of membrane proteins into defined lipid bilayers. Here, we describe an automated technique for reconstitution of membrane proteins into lipid bilayer membranes, which substantially reduces both the reconstitution time and the amount of protein required for the membrane formation. The method allows the investigation of single protein channels as well as insertion of Multiple Copies (similar to 10(7)) into a single bilayer. Despite a comparatively large membrane area (up to 300 mu m diameter), the high stability of the membrane permits the application of transmembrane voltages up to 300 mV. This feature is especially important for studies of inner membrane mitochondrial proteins, since they act at potentials up to similar to 200 mV under physiological conditions. It is a combination of these advantages that enables the detailed investigation of the minuscule single protein conductances typical for proton transporters. We have applied the new technique for the reconstitution and electrophysiological characterisation of human recombinant uncoupling protein 1, hUCP1, that has been overexpressed in E. coli and purified from inclusion bodies. We demonstrate that hUCP1 activity in the presence of fatty acids is comparable to the activity of UCP1 isolated from brown adipose tissue. (c) 2006 Elsevier B.V. All rights reserved.