甘油脱水酶α亚基基因的克隆、表达及纯化

Cloning and Expression of Glycerol Dehydratase α-Subunit Gene in E.coli and Purification of Expressed Product

目的克隆甘油脱水酶α亚基基因,构建表达载体,表达并纯化融合蛋白。方法采用PCR技术克隆甘油脱水酶α亚基gldA基因,并将其重组到含组氨酸标签的表达质粒pET-32a(+)中。将重组质粒经热激转化至大肠杆菌BL21(DE3)中,IPTG诱导表达。表达产物经金属螯合层析柱纯化,并进行Western blot分析及活性检测。结果重组表达质粒pET/gldA经酶切鉴定及序列分析,证明构建正确。转化E.coliBL21(DE3)后,重组蛋白获得可溶性表达,融合蛋白相对分子质量约81000,与预期大小一致。经Western blot分析,纯化蛋白能与6-Histidine抗体产生特异性免疫反应。α亚基经检测,未显示活性。结论已成功获得甘油脱水酶α亚基蛋白,为进一步研究其生物学性能奠定了基础。

Objective To clone glycerol dehydratase α-subunit gene,eonstruet expression vector and purify the expressed fusion protein. Methods Clone glycerol dehydratase α-subunit gldA gene by PCR and insert into expression vector pET-32a（ ＋ ）. Transform the construeted reeombinat plasmid to E. coli BL21 （DE3） by heat shock transformation for expression under induction of IPTG. The expressed product was purified by metal chelate chromatography, identified by Western blot and determined for activity. Results Both restriction analysis and sequencing result proved that recombinant plasmid pET/gldA was correctly constructed. The relative molecular mass of expressed soluble fusion protein was about 81 000, which was consistent with that expected. Western blot showed specific reaction of purified fusion protein with antibody against 6-Histidine. However, the protein showed no activity. Conclusion Glycerol dehydratase α-Subunit was successfully expressed, which laid a foundation of further study on its biological property.