Ni Dongchun, Huang Yihua
National Laboratory of Biomacromolecules, National Center of Protein Science-Beijing, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
Methods Mol Biol. 2015;1329:169-78. doi: 10.1007/978-1-4939-2871-2_13.
In gram-negative bacteria, assembly of outer membrane proteins requires the multicomponent β-barrel assembly machinery (BAM) complex, of which BamA is an essential and evolutionarily conserved integral outer membrane protein. To understand how BamA facilitates outer membrane protein biogenesis, it is important to obtain sufficient amounts of purified recombinant BamA protein for in vitro functional analysis and structure determination. In this chapter, we describe the protocol that we used in our laboratory for the cloning, expression, and purification of E. coli BamA and its N-terminal deletion variants for in vitro functional studies and for structure determination of the β-barrel domain alone (residues 426-810).
在革兰氏阴性菌中，外膜蛋白的组装需要多组分β-桶组装机制（BAM）复合体，其中BamA是一种必需的且在进化上保守的外膜整合蛋白。为了了解BamA如何促进外膜蛋白生物合成，获得足够量的纯化重组BamA蛋白用于体外功能分析和结构测定非常重要。在本章中，我们描述了我们实验室用于克隆、表达和纯化大肠杆菌BamA及其N端缺失变体的方案，用于体外功能研究和单独β-桶结构域（残基426 - 810）的结构测定。
