High-level expression of the mycobacterial porin MspA in Escherichia coli and purification of the recombinant protein
High-level expression of the mycobacterial porin MspA in Escherichia coli and purification of the recombinant protein
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DOI：
10.1016/s1570-0232(03)00130-2
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发表时间：
2003-06-25
影响因子：
3
通讯作者：
Niederweis, M
中科院分区：
文献类型：
--
作者：
Heinz, C;Karosi, S;Niederweis, M
MspA是一类新的四聚体孔蛋白家族的原型，为亲水性化合物通过耻垢分枝杆菌的外膜提供了主要的一般扩散途径。由于缺乏纯蛋白质，结构分析受到阻碍。用大肠杆菌中高水平表达的密码子替换mspA基因中富含gc的密码子后，成熟的mspA蛋白在大肠杆菌中过量产生。重组MspA （rMspA）单体（m - r20 000）通过阴离子交换和疏水相互作用层析纯化，每升培养物得到2.6 mg纯蛋白。这比天然蛋白的产量高出10倍。圆二色性显示rMspA折叠成类似天然的结构。在大肠杆菌中表达和体外纯化后，rMspA部分组装成通道形成的四聚体。因此，在大肠杆菌中过表达和色谱纯化是实现MspA高分辨率结构的关键步骤。(C) 2003 Elsevier Science B.V.版权所有
MspA is the prototype of a new family of tetrameric porins and provides the main general diffusion pathway for hydrophilic compounds through the outer membrane of Mycobacterium smegmatis. Structural analysis was hampered by the scarce amount of pure protein. After replacement of the GC-rich codons of the mspA gene by codons optimal for high-level expression in Escherichia coli, the mature MspA protein was overproduced in E. coli. The recombinant MspA (rMspA) monomer (M-r 20 000) was purified by anion exchange and hydrophobic interaction chromatography yielding 2.6 mg pure protein per liter of culture. This exceeded the yield of the native protein 10-fold. Circular dichroism revealed that rMspA is folded in a native-like structure. rMspA assembled partially to the channel-forming tetramer both during expression in E. coli and after purification in vitro. Thus, overexpression in E. coli and chromatographic purification are key steps towards a high resolution structure of MspA. (C) 2003 Elsevier Science B.V. All rights reserved.