Cloning,expression,purification and crystallization of murein-tripeptide amidase MpaA from Escherichia coli O157

doi:10.3969/j.issn.1000-1565.2016.04.013

Abstract

Abstract: 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.

Key words: Escherichia coli O157, peptidoglycan hydrolase, MpaA, crystal

CLC Number:

MA Yinliang,LIU Shuang,CUI Yaqi,KANG Xianjiang,LIU Xiuhua. Cloning,expression,purification and crystallization of murein-tripeptide amidase MpaA from Escherichia coli O157[J]. Journal of Hebei University (Natural Science Edition), 2016, 36(4): 412-416.

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