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Expression, Purification of Mycoplasma Genitalium Recombinant Protein MG427 and Studies on It’s Antioxidant Function

Author: ZhouJun
Tutor: WuYiMou；ZengHua
School: Nanhua University
Course: Pathogen Biology
Keywords: Mycoplasma genitalium MG427 hydrogen peroxide activity
CLC: R346
Type: Master's thesis
Year: 2011
Downloads: 19
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Abstract


Objective: Mycoplasma genitalium （M.genitalium） recombinant protein MG427 was expressed and it`s peroxidase activity was studied in order to further explore the possible mechanisms of the persistent infection of M.genitalium in the host.Methods: mg427 gene sequences were obtained from Genbank and specific primers were designed by Primer Premier 5.0. mg427 gene was then amplified by polymerase chain reaction （PCR） used M. genitalium strain G37 DNA for the template. The PCR products were directly cloned into pGEX-6p-1 expression vector to constructed recombinant plasmid pGEX-6p-1/mg427. A codon TGA in the position of 289～291 of gene was muted into TGG by site-directed mutagenesis. The mutagenized recombinant plasmid was transformed into E.coli BL21.The recombinant protein was expressed by the induction of IPTG, and analyzed by SDS-PAGE and Western blotting. The GST tag was removed by PreScission Protease after the recombinant protein was purified by GST affinity chromatography. The protein concentration was detected by bicinchoninic acid （BCA）. And then the ferrous oxidation xylenol orange （FOX） and DTT oxidation experiment were performed to assay the peroxide activity of MG427.Results: The target gene successfully amplified. The results of PCR and sequencing showed that the amplified DNA sequences were consistent with the sequences of the mg427 gene in GenBank. The TGA in position of 289～291 of mg427 gene was successly mutated into TGG. by PCR-mediated site-directed mutagenesis. SDS-PAGE analysis showed that the recombinant protein, exist mainly in soluble form, was successfully expressed and it`s relative molecular mass （Mr） was about 41.1 KDa. The recombinant protein was purified by GST affinity chromatography and then identificated by Western blotting. The GST tag in recombinant MG427 （rMG427） was successfully removed by PreScission Protease. FOX and DTT oxidation experiments showed that MG427 can degrade H2O2 in some degree and the different concentrations of MG427 had different speeds of degradeing H2O2. Cysteine exists in MG427 and disulfide bonds were formed in MG427, which involved in the peroxide metabolism.Conclusion:1., The prokaryotic expression recombinant pGEX-6p-1/MG427 was successfully constructed, and the protein with a molecular weight about 41.1 KDa was expressed in souble form in E.coli.2. MG427 has peroxide activity, in which the cysteine were involved in peroxide metabolism.

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