采用分子克隆方法将来源于大肠杆菌O157的mpaA基因构建入pET-21b(+)表达载体.诱导MpaA蛋白在大肠杆菌BL21(DE3)感受态细胞中高效表达,通过Ni亲和层析、离子交换层析和凝胶过滤层析三步法纯化得到高纯度蛋白.使用气象扩散法进行蛋白质晶体生长的初步筛选与优化,得到MpaA蛋白质单晶,并使用X射线衍射仪进行衍射数据的收集与处理.MpaA晶体分辨率为0.26 nm,属于P31空间群,晶胞参数为a=5.989 3 nm,b=5.989 3 nm,c=12.987 0 nm,β=90.000 °.MpaA晶体衍射数据的收集为后续结构解析和催化机制的阐明提供了基础.
The gene mpaA from Escherichia coli O157 was inserted into pET-21b(+)vector using molecular cloning method.The recombinant protein MpaA was overexpressed in E.coli BL21(DE3)competent cells,and purified to homogeneity sequentially via a three-step protocol consisting of Ni-chelating affinity column,ion-exchange column and size-exclusion column chromatography.Crystallization search and optimization for MpaA were carried out using the sitting-drop vapour-diffusion method.The MpaA crystal diffracted to 0.26 nm resolution and belonged to the space group P31,with unit-cell parameters a=5.989 3 nm,b=5.989 3 nm,c=12.987 0 nm and β=90.000 °.Preliminary crystallographic analysis builds up a good foundation for the structural determination and catalytic mechanism of MpaA.
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