Heinz Christian, Karosi Stefanie, Niederweis Michael
Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, D-91058, Erlangen, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Jun 25;790(1-2):337-48. doi: 10.1016/s1570-0232(03)00130-2.
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) 20000) 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.
MspA是一种新型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。由于纯蛋白数量稀少，结构分析受到阻碍。在用对大肠杆菌高水平表达最优的密码子替换mspA基因富含GC的密码子后，成熟的MspA蛋白在大肠杆菌中过量表达。重组MspA（rMspA）单体（分子量20000）通过阴离子交换和疏水相互作用色谱法纯化，每升培养物可产生2.6毫克纯蛋白。这比天然蛋白的产量高出10倍。圆二色性表明rMspA折叠成类似天然的结构。rMspA在大肠杆菌中表达期间以及体外纯化后都部分组装成形成通道的四聚体。因此，在大肠杆菌中过表达和色谱纯化是获得MspA高分辨率结构的关键步骤。
