内含肽融合标签介导的人乙醛脱氢酶2基因原核表达

Expression of recombinant human aldehyde dehydrogenase 2 by using intein induced expression system

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。

To achieve the recombinant human aldehyde dehydrogenase 2 proteins with high activity, the ALDH2 gene was amplified by PCR. The cloned and expressed plasmids pGEM-T-ALDH2 and pTYB11-ALDH2 were constructed. The recombinant expression plasmids was induced by IPTG. Through orthogonal experiment, the results showed that the optimal strategy was the expression conditions (OD 0.3, 1.0 mmol·L-1 IPTG, 30 ℃, 4 h). Because all the target proteins were almost expressed in the form of inclusion body, the bioactive acetaldehyde dehydrogenase was obtained by means of degeneration and renaturation for the inclusion body. Using the IMPACTTM-CN protein purification system, the similar natural ALDH2 protein was obtained from the self cleavage of fused intein protein, and the protein concentration was 0.045 mg·mL-1 by the Bradford method. The activity of purification protein was 462 U·mg-1.