Expression, Purification, and Screening of BamE, a Component of the BAM Complex, for Structural Characterization.
Expression, Purification, and Screening of BamE, a Component of the BAM Complex, for Structural Characterization.
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BamE（BAM 复合物的一个组成部分）的表达、纯化和筛选，用于结构表征。
DOI：
10.1007/978-1-4939-2871-2_19
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Jeeves M
中科院分区：
文献类型：
--
作者：
Jeeves M
在革兰氏阴性菌中，整体外膜β-桶蛋白（OMP）由β-桶组装机复合体（BAM复合体）组装。该复合物包括必需成分BamA，一个由羧基末端β桶结构域和五个多肽转运相关结构域（POTRA）组成的OMP，以及脂蛋白BamA。在大肠杆菌中，该复合物含有额外的三种脂蛋白，BamB， C和E，这是将omp有效递送到外膜所必需的。在这里，我们提供了用于研究目的的BamE的生产，同位素标记，纯化和功能筛选的方法。使用渗透冲击、Ni-NTA纯化和尺寸排除色谱等技术描述了可溶性和野生型膜系链形式的BamE的纯化策略。功能筛选使用一个简单的板试验也描述，允许筛选外膜渗透性缺陷。
In Gram-negative bacteria, integral outer membrane β-barrel proteins (OMP) are assembled by the β-barrel assembly machine complex, or BAM complex. This complex includes the essential components BamA, an OMP composed of a carboxyl terminal β-barrel domain and five polypeptide transport-associated domains (POTRA), and the lipoprotein BamD. InEscherichiacoli, the complex contains an additional three lipoproteins, BamB, C and E required for efficient delivery of OMPs to the outer membrane. Here we provide methods for production, isotope labeling, purification, and functional screening of BamE for research purposes. Purification strategies of both the soluble and wild-type membrane-tethered forms of BamE are described using techniques including osmotic shock, Ni-NTA purification, and size-exclusion chromatography. Functional screening using a simple plate assay is also described which allows screening for defects in outer membrane permeability.