嗜碱耐盐芽孢杆菌乙醛脱氢酶基因aldC的克隆及组氨酸融合酶蛋白的表达

Cloning of Aldehyde Dehydrogenase Gene aldC and Expression of Bacillus halodurans XJU-1

应用PCR扩增嗜碱耐盐芽孢杆菌乙醛脱氢酶基因aldC,重组至原核表达载体pET30b(+),转化大肠杆菌BL21(DE3),诱导表达的重组乙醛脱氢酶在碳端含有一个组氨酸标签。SDS-PAGE考察蛋白的表达量和亚基分子量,并对Km值进行了考察。成功构建了pET30b-aldC原核表达质粒;诱导表达了亚基分子量约为56 kDa的组氨酸融合蛋白;乙醛脱氢酶活性比对照菌增加了约3.5倍;Km值为2.874 mmol/L。成功克隆并诱导表达了含组氨酸标签的嗜碱耐盐芽孢杆菌乙醛脱氢酶基因aldC。

To clone aldehyde dehydrogenase gene aldC from Bacillus halodurans XJU-1 and express aldC fusion proteins with a His6-tag,aldC gene without terminator was amplified by PCR and cloned into plasmid pET30b（＋）.The recombinant plasmid was transformed into E.coli BL21（DE3）.The transformant colony was then induced with IPTG and expressed a fusion proteins with 6×His at C terminus.The target protein was analysed by SDS-PAGE and Km values was investigated.The gene aldC was amplified and cloned into plasmid pET30b（＋） successfully.The molecular weight of the fusion enzyme as estimated by SDS-PAGE was approximately 56,000.The enzyme activity was 3.5 times than the contrast bacteria.The Km values of the enzyme were 2.874mM for acetaldehyde.The recombinant plasmid was successfully constructed and the aldehyde dehydrogenase gene was expressed with His6 tag in E.coli BL21（DE3）.