Direct transfer of membrane proteins from bacteria to planar bilayers for rapid screening by single-channel recording
Direct transfer of membrane proteins from bacteria to planar bilayers for rapid screening by single-channel recording
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DOI：
10.1038/nchembio793
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发表时间：
2006-06-01
影响因子：
14.8
通讯作者：
Bayley, Hagan
中科院分区：
文献类型：
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作者：
Holden, Matthew A.;Jayasinghe, Lakmal;Bayley, Hagan
虽然检测平面双层中的膜蛋白是评估其药理学和生理作用的有力方法，但将膜蛋白引入双层通常是一个困难的过程(1)。在这里，我们使用机械探针将膜蛋白直接从大肠杆菌表达菌落转移到人工脂质双分子层。通过这种方式，单通道电记录可以从两类主要的膜蛋白，α -螺旋束和β桶，分别由K+通道和细菌成孔毒素表示。此外，我们还检测了双组分毒素杀白细胞素（Luk），它由LukF和LukS亚基组成。我们将分别表达LukF和luks的菌落混合，并将混合物转移到平面双层上，从而产生功能性的Luk孔。通过这种方法，我们快速筛选了突变Luk亚基的二元组合，以获得特定功能：结合分子接头的能力。我们认为，从细胞到双层的直接转移将在膜蛋白质组学和传感器阵列的构建的几个方面有用。
Although the examination of membrane proteins in planar bilayers is a powerful methodology for evaluating their pharmacology and physiological roles, introducing membrane proteins into bilayers is often a difficult process(1). Here, we use a mechanical probe to transfer membrane proteins directly from Escherichia coli expression colonies to artificial lipid bilayers. In this way, single-channel electrical recordings can be made from both of the major classes of membrane proteins, alpha-helix bundles and beta barrels, which are represented respectively by a K+ channel and a bacterial pore-forming toxin. Further, we examined the bicomponent toxin leukocidin (Luk), which is composed of LukF and LukS subunits. We mixed separate LukF- and LukS-expressing colonies and transferred the mixture to a planar bilayer, which generated functional Luk pores. By this means, we rapidly screened binary combinations of mutant Luk subunits for a specific function: the ability to bind a molecular adaptor. We suggest that direct transfer from cells to bilayers will be useful in several aspects of membrane proteomics and in the construction of sensor arrays.