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| 谷胱甘肽S-转移酶的诱导表达及提纯 |
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杨丹1,叶佩莹2,万津1,刘永平1,马辉文1
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1. 武汉大学药学院,湖北武汉,430072;2. 武汉大学生命科学学院,湖北武汉,430072
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| 摘要: |
| 含有日本血吸虫谷胱甘肽S-转移酶基因的质粒pGEX-5X-1在大肠杆菌BL21(DE3)菌株中得到表达.37℃[1]下用1% 乳糖诱导谷胱甘肽S-转移酶(GST)的最适表达时间为 3h.盐析粗提酶液并以Habig法[2]监测GST的活性,用pH6.0,饱和度为30%硫酸铵去除杂蛋白,并调pH7.5,饱和度 70%硫酸铵使GST大量析出并保持活性.用透析法脱盐,并用Sephadex-G50凝胶对GST进行了初步纯化.初步探讨了GST的保存方法. |
| 关键词: 谷胱甘肽S-转移酶 基因表达 GST的纯化 |
| DOI： |
| 分类号:Q814 |
| 基金项目: |
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| Expression and Purification of Glutathione S-Transferase |
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| Abstract: |
| Glutathione S-transferase (GST)gene of Schistosoma japonicum harboring in the plasmid pGEX-5X-1 was expressed in Escherichia coli BL21(DE3) induced with lactose. The optimal inducing time was 3 hours at the temperature of 37℃. The expressed GST was salted out by 70% saturated ammonium sulphate, dialysed and partially purified with Sephadex-G50 gel filtration-chromatography. The enzymatic activity of GST in the cell lysate was determined with spectrophotometericrely described by Habig et al. and the expressing level of GST evaluated with SDS-PAGE was used as indexes to optimize the process of induction and purification of GST. |
| Key words: glutathione S-transferase gene expression purification of GST |
