Journal of Hebei University(Natural Science Edition) >

2016 , Vol. 36 >Issue 4: 412 - 416

DOI: https://doi.org/10.3969/j.issn.1000-1565.2016.04.013

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

  • MA Yinliang ,
  • LIU Shuang ,
  • CUI Yaqi ,
  • KANG Xianjiang ,
  • LIU Xiuhua
Expand
  • College of Life Sciences, Hebei University, Baoding 071002, China

Received date: 2015-10-16

  Online published: 2016-07-25

Fold

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

Cite this article

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 . DOI: 10.3969/j.issn.1000-1565.2016.04.013

References

[1] WALDEMAR V,BERNARD J,PAULETTE C,et al.Bacterial peptidoglycan(murein)hydrolases [J].FEMS Microbiol Rev,2008a,32:259-286.DOI:10.1111/j.1574-6976.2007.00099.x.
[2] WALDEMAR V,DIDIER B,MIGUEL A D P.Peptidoglycan structure and architecture [J].FEMS Microbiol Rev,2008b,32:149-167.DOI:10.1111/j.1574-6976.2007.00094.x.
[3] HEIJENOORT J V.Peptidoglycan hydrolases of escherichia coli [J].Microbiol Mol Biol,2011,75(4):636-663.DOI:10.1128/mmbr.00022-11.
[4] ANANTHARAMAN V,ARAVIND L.Evolutionary history,structural features and biochemical diversity of the NlpC/P60 superfamily of enzymes [J].Genome Biology,2003,4:R11.DOI:10.1186/gb-2003-4-2-r11.
[5] JAMES T P,TSUYOSHI U.How bacteria consume their own exoskeletons(Turnover and recycling of cell wall peptidoglycan)[J].Microbiol Mol Biol,2008,72(2):211-227.DOI:10.1128/mmbr.00027-07.
[6] JACOBS C,HUANG LI-JUN,BARTOWSKY E,et al.Bacterial cell wall recycling provides cytosolic muropeptides as effectors for beta-lactamase induction [J].The EMBO Journal,1994,13(19):4684-4694.
[7] TSUYOSHI U,JAMES T P.Identification of MpaA,an amidase in Escherichia coli that hydrolyzes the gamma-D-glutamyl-meso-diaminopimelate bond in murein peptides [J].Journal of Bacteriology,2003,185(2):679-682.DOI:10.1128/jb.185.2.679-682.2003.
[8] SCHROEDER U,HENRICH B,FINK J,et al.Peptidase D of Escherichia coli K-12,a metallopeptidase of low substrate specificity [J].FEMS Microbiology Letters,1994,123(1-2):153-159.DOI:http://dx.doi.org/10.1111/j.1574-6968.1994.tb07215.x.
[9] GULICK A M,SCHMIDT D M Z,GERLT JOHN A,et al.Evolution of enzymatic activities in the enolase superfamily:crystal structures of the L-Ala-D/L-Glu epimerases from Escherichia coli and Bacillus subtilis [J].Biochemistry,2001,40(51):15716-15724.DOI:10.1021/bi011641p.
[10] MAQBOOL A,HERVE M,LECREULX D M,et al.MpaA is a murein-tripeptide-specific zinc carboxypeptidase that functions as part of a catabolic pathway for peptidoglycan-derived peptides in gamma-proteobacteria [J].Biochem J,2012,448:329-341.DOI:10.1042/BJ20121164.
[11] OTWINOWSKI Z,WLANEK M.Processing of X-ray diffraction data collected in oscillation mode [J].Methods Enzymol,1997,276:307-326.DOI:10.1016/S0076-6879(97)76066-X.
Options
Outlines

/

〈 〉