小鼠核糖核酸酶抑制剂异源高效可溶性表达、纯化及活性研究
Efficient Soluble Expression,Purification and Activity of Murine Ribonuclease Inhibitor
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摘要: 为获得高效可溶性表达的小鼠核糖核酸酶抑制剂(MRNI),本文通过构建含SUMO、IF2、GST、NusA、MsyB、Trx和 MBP融合标签的重组表达载体,以大肠杆菌BL21(DE3)作为宿主菌进行自诱导(auto-induction,AI)表达,利用MagNi磁珠检测及电泳分析MRNI的表达状况,并对其培养温度及时间进行优化,同时,与其他公司核糖核酸酶抑制剂(RI)活性进行对比。结果表明,重组菌BL21(DE3)(pNBEⅡ-MRNI)诱导表达的可溶性融合蛋白IF2-MRNI的产量最高,最适诱导条件为37 ℃,诱导培养6 h,20 ℃培养24 h。磁珠纯化后获得的融合蛋白IF2-MRNI浓度为3621.3 mg/L,检测其酶活性约为40 U/μL。该酶具有抑制RNase A活性,防止RNA被降解的作用,为RI的生产及应用提供理论基础。Abstract: The paper was to obtain efficient soluble expression of mouse ribonuclease inhibitor(MRNI). By constructing a recombinant expression vector containing SUMO,IF2,GST,NusA,MsyB,Trx and MBP fusion tags,E. coli BL21(DE3)was used as a host strain for auto-induction(AI)expression,and MagNi magnetic beads were used for detection. The expression of MRNI was analyzed by electrophoresis,and the culture temperature and time were optimized. The results showed that the soluble fusion protein IF2-MRNI induced by recombinant BL21(DE3)(pNBEⅡ-MRNI)had the highest yield,and the optimal induction condition was 37 ℃,induced for 6 h,and cultured at 20 ℃ for 24 h. The concentration of the fusion protein IF2-MRNI obtained after purification of the magnetic beads was 3621.3 mg/L. Compared with RI activity of other companies,the enzyme activity was about 40 U/μL. The enzyme had the function of inhibiting RNase A activity and preventing degradation of RNA,and provided a theoretical basis for the production and application of RI.
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