双顺反子翻译偶联表达载体的构建及蚓激酶基因F238在大肠杆菌中的可溶性表达

Construction of translation-coupled bicistronic expression vector and soluble expression of lumbrokinase F238 in E. coli

摘要:

为了实现外源蛋白在大肠杆菌中的可溶性表达, 利用硫氧还蛋白作为分子伴侣构建双顺反子翻译偶联表达载体pDICT。将大肠杆菌硫氧还蛋白基因插入到pET22b载体Nde I和EcoR I位点之间, 同时在硫氧还蛋白编码基因的终止密码子前加入核糖体结合位点, 构建成双顺反子翻译偶联表达载体pDICT。将蚓激酶基因F238克隆到该载体, 转化大肠杆菌BL21(DE3)并诱导表达。SDS-PAGE结果表明, 所表达的蚓激酶F238是可溶性蛋白。利用血纤维蛋白法对表达产物进行活性测定, 重组蚓激酶F238不仅具有纤溶酶活性, 而且具有激活纤溶酶原的激酶活性。该双顺反子翻译偶联表达载体的构建, 为在大肠杆菌中可溶性表达外源蛋白提供了新方法。

关键词:蚓激酶, 分子伴侣, 双顺反子, 可溶性表达

Abstract:

In order to express soluble foreign proteins in E. coli, a bicistronic translation-coupled expression vector pDICT was constructed by using thioredoxin as a molecular chaperone. In this study, the expression vector pDICT was built by inserting thioredoxin gene into the pET22b vector between Nde I and EcoR I sites, and adding ribosome binding site before the stop codon of thioredoxin. Lumbrokinase gene F238 was cloned into this vector, and transformed into E. coli BL21(DE3), then the recombinant protein was induced by IPTG. SDS-PAGE results showed that the Lumbrokinase F238 was soluble expressed. The fibrinolytic activity was measured by using artificial fibrin plates, and the result indicated that Lumbrokinase could directly dissolve fibrous protein, and also had fibrinokinase activity which could indirectly dissolve fibrous protein. Construction of the bicistronic translation-coupled expression vector provides a new method to achieve soluble expression of heterologous proteins in E. coli.

Key words:Lumbrokinase, Molecular chaperones, Bicistronic, Soluble expression