| 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 |
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| 引用本文： | 付大伟, 孙莹莹, 徐伟. 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[J]. 食品工业科技, 2019, 40(10): 174-178,183. DOI: 10.13386/j.issn1002-0306.2019.10.029 |
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| 作者姓名： | 付大伟 孙莹莹 徐伟 |
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| 作者单位： | 1.哈尔滨商业大学食品科学与工程重点实验室, 黑龙江哈尔滨 150076 |
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| 基金项目： | 哈尔滨商业大学博士科研启动基金项目(92508633) |
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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。
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| 关 键 词： | 核糖核酸酶抑制剂 融合标签 诱导表达 磁珠纯化 |
| 收稿时间： | 2018-08-29 |
Efficient Soluble Expression,Purification and Activity of Murine Ribonuclease Inhibitor |
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| FU Da-wei, SUN Ying-ying, XU Wei. Efficient Soluble Expression,Purification and Activity of Murine Ribonuclease Inhibitor[J]. Science and Technology of Food Industry, 2019, 40(10): 174-178,183. DOI: 10.13386/j.issn1002-0306.2019.10.029 |
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| Authors: | FU Da-wei SUN Ying-ying XU Wei |
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| Affiliation: | 1.Key Laboratory for Food Science and Engineering, Harbin University of Commerce, Harbin 150076, China |
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| 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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| Keywords: | ribonuclease inhibitor fusion tags inducible expression magnetic bead purification |
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