A systematic assessment of mature MBP in membrane protein production: overexpression, membrane targeting and purification.
A systematic assessment of mature MBP in membrane protein production: overexpression, membrane targeting and purification.
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DOI：
10.1016/j.pep.2011.06.001
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发表时间：
2011-11
影响因子：
1.6
通讯作者：
Cross, Timothy A.
中科院分区：
文献类型：
--
作者：
Hu, Jian;Qin, Huajun;Gao, Fei Philip;Cross, Timothy A.
获得足够的天然或类天然状态的膜蛋白仍然是膜蛋白结构生物学中的一个挑战。麦芽糖结合蛋白（MBP）已被广泛用作提高膜蛋白产量的融合伴侣。在目前的工作中，对 42 种膜蛋白应用成熟 MBP (mMBP) 进行膜蛋白过表达和纯化进行了系统评估，其中大多数膜蛋白在与 N 末端 Histag 融合的膜组分中没有表达或表达水平较低。结果发现，使用 MBP 时，大多数小膜蛋白在大肠杆菌的天然膜中过度表达。此外，融合体的蛋白水解是在膜上进行的，无需用去垢剂溶解，从而开发出一种有效的方案，通过一步亲和层析直接从膜组分中纯化目标膜蛋白。我们的结果表明，mMBP 是小膜蛋白过表达、膜靶向和纯化的极佳融合伴侣。本表达和纯化方法可能是结构和功能研究中大规模制备小膜蛋白的良好解决方案。
Obtaining enough membrane protein in native or native-like status is still a challenge in membrane protein structure biology. Maltose binding protein (MBP) has been widely used as a fusion partner in improving membrane protein production. In the present work, a systematic assessment on the application of mature MBP (mMBP) for membrane protein overexpression and purification was performed on 42 membrane proteins, most of which showed no or poor expression level in membrane fraction fused with an N terminal Histag. It was found that most of the small membrane proteins were overexpressed in the native membrane of E. coli when using MBP. In addition, the proteolysis of the fusions were performed on the membrane without solublilization with detergents, leading to the development of an efficient protocol to directly purify the target membrane proteins from the membrane fraction through a one-step affinity chromatography. Our results indicated that mMBP is an excellent fusion partner for overexpression, membrane targeting and purification of small membrane proteins. The present expression and purification method may be a good solution for the large scale preparation of small membrane proteins in structural and functional studies.
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影响因子：
8
作者：
Korepanova, A;Gao, FP;Cross, TA
通讯作者：
Cross, TA
影响因子：
64.8
作者：
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影响因子：
1.6
作者：
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通讯作者：
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影响因子：
1.6
作者：
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通讯作者：
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影响因子：
64.5
作者：
Ulbrandt, ND;Newitt, JA;Bernstein, HD
通讯作者：
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