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3-甾酮-Δ~1-脱氢酶的融合表达及纯化 被引量:1

Fused Expression and Purification of 3-Ketosteroid-Δ^(1)-Dehydrogenase
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摘要 3-甾酮-Δ1-脱氢酶是甾体母核降解的关键酶,能催化3-甾酮化合物在C1,2位处脱氢,提高原有底物的生物活性。利用表达载体pET-30a,实现了简单节杆菌3-甾酮-Δ1-脱氢酶在大肠杆菌BL21(DE3)中的高效表达。利用SDS-PAGE分别对超声破碎后的上清和沉淀进行分析,并用分光光度法检测酶的活力。结果表明,表达出的蛋白主要以无活性的包涵体形式存在,利用Ni离子螯合层析的方法纯化融合蛋白,复性后的酶活比简单节杆菌提高了近10倍。 3-Ketosteroid-△^1-dehydrogenase is the key enzyme in the degradation of nucleus of steroids, which can introduce a double bond into the position C1.2. The dehydrogenated product has much higher biologic activity than the original substrate. In this research, the gene encoding 3-ketoste throbacter simplex was cloned into expression vector pET-30a constituting which was transformed into BL21(DE3). Positive transformant was cultivated to induce expression of 3-ketosteroid-△^1-dehydrogenase. Inducting for 4 h, th roid-△^1-dehydrogenase recombination vector and IPTG was added i from Arthrobacter simplex was cloned into expression vector pET-30a constituting recombination vector pET-ksdD nto culture e supernatants and sedimentation collected after sonic disruption were analyzed by SDS-PAGE. The result indicated 3-ketosteroid-△^1dehydrogenase was expressed in E. coli successfully. The activity result assayed by spectrophotometrical method and in vitro transformation test indicated that the expressed protein was in form of insoluble bodies. Purified step was carried out in the denatured condition using His-Bind purification kit. The activity of recovered enzyme was about 10 folders of that of the extracts from Arthrobacter simplex.
出处 《化学与生物工程》 CAS 2006年第9期34-37,共4页 Chemistry & Bioengineering
关键词 3-甾酮-△^1-脱氢酶 融合表达 包涵体 转化 3-ketosteroid-△^-1-dehydrogenase fused expression insoluble body transformation
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