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结核分支杆菌katG蛋白的高表达与纯化
http://www.100md.com 2004年8月15日
     张文宏 翁心华 季朝能 毛裕民 陈一平 陈澍 邬祥惠 2004-7-31 17:33:00 中华传染病杂志 2000年第1期第18卷

    【摘要】目的 表达与纯化结核分支杆菌katG蛋白,为深入研究异烟肼耐药机制奠定基础。方法 将含有katG基因的pET24b-katG表达载体转化大肠杆菌BL21(DE3) 菌株,在异丙基硫代-β-D-半乳糖苷(IPTG)诱导下表达,分别对不同诱导时间的表达产 物进行SDS-PAGE以及考马斯亮蓝染 色。获得稳定的高表达菌株后采用Xpress TM蛋白纯化系统对超声破菌液进行纯 化。最后对纯化产物进行过氧化氢酶活性的初步检测。结果 对诱导后的重组大肠杆菌菌液进行十二烷基硫酸钠-聚丙烯酰胺凝 胶电泳(SDS-PAGE)以及考马斯亮蓝染色后 发现相对分子质量约为80 000。表达蛋白量约占总蛋白量的17.7%。对重组katG基因表达产 物进行 纯化的结果发现,以350 mmol/L咪唑洗脱时的纯化效果最理想,蛋白纯度可达90%以上。对 表达产物进行过氧化氢酶活性初步检测证明,重组的katG基因产物具有过氧化氢酶活性。[ HTH〗结论 通过pET24b-katG表达质粒转化大肠杆菌可获得基因重组的katG高表 达菌株,表达产物具有一定酶活性,经纯化后可达到较高的纯度。

    Overexpression and purification of catalase-peroxidase katG from mycobacterium tuberculosis

    ZHANG Wenhong WENG Xinhua JI Chaoneng et al.

    (Departme nt of Infect ious Diseases, Huashan Hospital, Shanghai Medical University, Shanghai 200040, China)

    【Abstract】Objective To express and purify the catala se-peroxidase katG gene from mycobacterium tuberculosis. Methods Plasmid pET24b containing katG was transferred into com petent Escherichia coli and katG gene was overexpressed by the challenge of isopropylthio-β-D-glactoside(IPTG). The expression of katG protein was on e-step purified by Xpress systemTM. Furthermore, the catalase activity of katG protein was preliminarily detec ted. Results The recombinant escherichia coli produced katG p rotein in large quantities, accounting for 17.7% of total cell protein. The mole cular mass of katG protein was estimated to be 80 000 by sodium dodecyl sulfate -polyacrylamide gradient gel electrophresis (SDS-PAGE). It was found that imidazole at the concentration of 350 mmol/L could elute the katG protein most efficiently and yield the final preparation at greater than 90% purity. Th e katG protein was preliminarily detected to have the activity of catalase. Conclusions The stable katG overexpressing recombinant Escherichi a coli can be constructed by the plasmid pET24b containing katG gene. The reco mbinant strain can produce katG protein with catalase activity and the product o f which can be purified into higher activity. ......

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