【摘要】 从Bacillus sp.110-2基因组上分离得到的耐碱锰超氧化物歧化酶基因(Mn-sodA),将其连接到原核表达载体pET-30a(+),得到重组载体pET-sodA,重组载体在大肠杆菌(Escherichia coli)BL21(DE3)中经异丙基硫代-β-D-半乳糖苷(IPTG)诱导得到表达。SDS-PAGE分析显示,融合表达产物的分子量大小为26.5kD。对含有sodA基因工程菌的表达情况研究表明,重组蛋白全部以可溶性形式存在;重组酶的比活力252U/mg,为原始菌株的2.1倍,目的基因在大肠杆菌中实现了高效表达;而且,重组酶还保留了野生型酶强的耐碱能力。BL21(pET-sodA)基因工程菌的构建提供了一种可利用的耐碱蛋白资源,同时探索了一种极端酶的生产方法。更多还原

【Abstract】 The alkalotolerant Mn-superoxide dismutase encoding gene (Mn-sodA) from Bacillus sp. 110-2 was obtained, and inserted in pET-30a (+) to yield the recombinant expression vector pET-sodA. Over-expression of Mn-SOD in Escherichia coli BL21 (DE3) was achieved with pET-sodA, which was induced by Isopropyl-β-D-galactosidase (IPTG). SDS-PAGE analysis showed that an over-expressed recombinant product about 26.5 kD, consistent with the molecular weight predicted from gene sequence was produced. The recombinant protein was soluble in BL21(DE3). The specific activity of the recombinant enzyme Mn-SOD was 252 U/mg, which was 2.1 times than that of wild type. It indicated that the expression of Mn-sodA was high in E.coli. Furthermore, the recombinant en- zyme had a strong ability of alkali-resistance as wild enzyme. From the engineered strain-BL21 (pET-sodA), an alkali-resistance protein resources and a method to product the enzyme with extreme ability can be obtained.更多还原