Prokaryotic expression and activity analysis of 5′-methylthioadenosine nucleosidase in Mycobacterium tuberculosis
10.3760/cma.j.issn.0254-5101.2012.07.005
- VernacularTitle:结核分枝杆菌甲硫腺苷核苷酶的原核表达及活性分析
- Author:
Haizhen CHEN
;
Hua YANG
;
Zhongyi HU
;
Huansen YANG
;
Hui MA
;
Shihui GAO
;
Qi GUO
;
Wenjuan BAI
;
Lianhua QIN
;
Lianqing LI
- Publication Type:Journal Article
- Keywords:
Mycobacterium tuberculosis;
MTAN;
Rv0091;
Recombinant protein;
Enzyme activity analysis
- From:
Chinese Journal of Microbiology and Immunology
2012;32(7):589-594
- CountryChina
- Language:Chinese
-
Abstract:
Objective To clone and express of Rv0091 encoding protein in Mycobacterium tuberculosis,identify and characterize of the enzyme activities.Methods Construct the Rv0091 prokaryotic expression plasmid,the vector was transformed into E.coli strain BL21trxB.After induced by IPTG,recombinant protein was purified by Ni2+-NTA chromatography and analyzed for purity by SDS-PAGE gels stained with Coomassie Blue.Immunological activity was identified by Western blot.The recombinant protein molecular weight was identified by Mass spectrometry.The enzyme-coupled assay detectes enzyme activity.Results The expression plasmid pET32a-Rv0091 was constructed and expressed in E.coli.BL21trxB,and the optimum expression system was conformed.The purity of the recombinant protein was more than 95％.Western blot analysis confirmed that recombinant protein was one of Mycobacterium tuberculosis proteins.Mass spectrometry identified the relative molecular weight and theoretical molecular weight was basically the same.Enzyme assay showed the recombinant protein able to catalyze the substrate MTA.Enzymatic properties showed that the optimal buffer for the phosphate and Hepes buffer,the poor thermal stability of the enzyme,the optimal temperature of 37℃,optimal pH10-12,when the pH ≤7,the protein denaturation and loss of some vitality.Conclusion The recombinant protein methylthioadenosine nucleosidase(MTAN) was obtained and enzyme activity was detected and plays a key role in the metabolism of Mycobacterium tuberculosis.
