内含肽融合标签介导的人乙醛脱氢酶2基因原核表达

发布时间：2019-02-12 18:48

【摘要】：为获得高活性重组人乙醛脱氢酶2(ALDH2)重组蛋白,研究通过PCR获得ALDH2基因,先后构建克隆和表达载体p GEM-ALDH2和p TYB11-ALDH2。转化的阳性重组菌经IPTG诱导,SDS-PAGE显示融合蛋白获得大量表达。通过正交试验对表达条件优化设计,结果表明,诱导OD值0.3,30℃,IPTG浓度1.0 mmol·L-1,诱导表达4 h为最优表达条件。由于融合蛋白以包涵体形式存在,经变性、复性,使用IMPACTTM-CN蛋白纯化系统,经内含肽标签蛋白自裂解洗脱、纯化,获得仅含有几个载体氨基酸的ALDH2蛋白,蛋白浓度为0.045 mg·m L-1。纯化的ALDH2蛋白比活力为462 U·mg-1。
[Abstract]:In order to obtain recombinant human aldehyde dehydrogenase 2 (ALDH2) recombinant protein with high activity, the ALDH2 gene was obtained by PCR. The cloning and expression vectors of p GEM-ALDH2 and p TYB11-ALDH2. were constructed successively. The positive recombinant strain was induced by IPTG, and SDS-PAGE showed that the fusion protein was highly expressed. The expression conditions were optimized by orthogonal experiment. The results showed that the optimal expression conditions were induced OD value of 0.33 鈩，

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