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

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。

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.