3α-羟类固醇脱氢酶基因的质粒载体构建和表达

目的 实现3α-羟类固醇脱氢酶基因在大肠埃希菌中的高可溶性表达.方法 从土壤中分离睾丸酮丛毛单胞菌,提取其基因组DNA,PCR扩增3α-羟类固醇脱氢酶(3α-HSD)基因,将它克隆到原核表达载体上进行诱导表达.提取细菌总蛋白进行SDS-PAGE分析并测定酶活性.结果 经核苷酸序列测定和酶切鉴定结果表明,成功地构建了重组质粒,IPTG诱导表达后,获得融合蛋白,SDS-PAGE初步测定目的蛋白的相对分子量约为29kDa,与预期理论值一致;酶活性测定结果表明菌体可溶性总蛋白HSD酶比活性为142.81 U/mg,是对照BL21的12.97倍.结论 该研究成功地构建了3α-羟类固醇脱氢酶基因高效原核表达系统,为利用基因工程手段大量制备3α-HSD的工作奠定了基础.

Cloning and expression of 3o-hydroxysteroid dehydrogenase gene in E. coli

Objective To achieve the overexpression of 3α-hydroxysteroid dehydrogenase(3α-HSD) gene in E.coli and better enzyme activity of the expressed product. Method 3α-hydroxysteroid dehydrogenase gene was amplified by PCR from a wild Comamonas testosteroni. The PCR product was cloned into pUC19-T vector and sequenced. The 3α-HSD prokaryotic expression system was constructed with plasmid pET28α as the vector and E. coli BL21 as the host bacteria.The fusion protein with an N-terminal His-tag sequence was purified in one step using metal chelate affinity chromatography to homogeneity on a Ni2 + -Sepharose column. Result The SDS-PAGE analysis revealed that the molecular weight of the recombinant protein was about 29 kDa. The enzyme activity detection showed that the specific activity of the expressed product in the total soluble protein was 1428.49 U/mg,129.74 times of that of E. coliBL21. Conclusion The soluble expression of 3α-HSD gene in E. coli was successful.