缬氨酸转氨酶基因原核表达载体构建及表达

摘要

构建了具有缬氨酸转氨酶基因的大肠杆菌工程菌,对该酶表达条件进行了优化.PCR结果表明,扩增出一特异DNA条带且长度与avtA基因长度1 254 bp符合.通过纸层析检测,筛选到了阳性克隆,但是酶活偏低.SDS-PAGE凝胶电泳显示目的蛋白表达量较低.酶表达优化结果显示:蛋白胨浓度12 g/L,IPTG浓度0.4 mmol/L,经过8h诱导,酶活达到最大值.%The engineered strain of Escherichia coli with valine-pyruvate transaminase gene was constructed and the expression condition for valine-pyruvate transaminase was optimized. The result of PCR showed that a special DNA band was amplified and the length of the band was accord with the length of avtA, 1 254 bp. Activity of valine-pyruvate transaminase was found by paper chromatography but the enzyme activity was not high. The expression of valine-pyruvate transaminase was evaluated by SDS-PAGE and a high expression was displayed. The optimal conditions were peptone 12 g/L,IPTG 0. 4 mmol/L and induced time 8 h.