融合蛋白NusA-hRI的高效异源表达、纯化及活性分析

doi:10.13523/j.cb.20190304

中国生物工程杂志

2019, Vol. 39

Issue (3): 21-28 DOI: 10.13523/j.cb.20190304

研究报告

融合蛋白NusA-hRI的高效异源表达、纯化及活性分析

付大伟(

),孙莹莹,徐伟

哈尔滨商业大学食品工程学院哈尔滨 150076

Efficient Heterologous Expression, Purification and Activity Analysis of Fusion Protein NusA-hRI

Da-wei FU(

),Ying-ying SUN,wei XU

Key Laboratory for Food Science and Engineering, Harbin University of Commerce, Harbin 150076, China

全文: PDF(858 KB) HTML

摘要：

人核糖核酸酶抑制因子 (human ribonuclease inhibitor, hRI) 是一种能够调节核糖核酸酶活性的酸性包浆蛋白。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,从而使 hRI 的表达量得以提升。利用MagNi磁珠纯化及电泳分析hRI的表达状况,通过RNase/Sepharose亲和层析获得纯度较高的蛋白。纯化后获得的融合蛋白浓度为2 960.513mg/L,与其它公司的hRI活性进行比较,检测其酶活性约为50U/μl,并使其成功用于RNA的保护,为NusA-hRI的应用提供理论依据。

关键词： 人核糖核酸酶抑制因子; 融合标签; 诱导表达; 磁珠纯化; 亲和层析

Abstract:

Human ribonuclease inhibitor (hRI) is an acidic pulping protein that regulates ribonuclease activity. 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, thereby the expression level of hRI is improved. The expression of hRI was analyzed by MagNi magnetic bead purification and electrophoresis, and the protein with higher purity was obtained by RNase/Sepharose affinity chromatography.The concentration of the fusion protein obtained after purification was 2 960.513mg/L, which was compared with other companies, and its enzyme activity was about 50U/μl, which was successfully used for RNA protection. Purely provide a theoretic-al basis for the application of NusA-hRI.

Key words: Human ribonuclease inhibitor Fusion tags Auto-induction Magnetic bead me-thod Affinity chromatography

收稿日期: 2018-09-30 出版日期: 2019-04-12

ZTFLH:

Q816

通讯作者: 付大伟 E-mail: fudaweinovo@163.com

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引用本文:

付大伟,孙莹莹,徐伟. 融合蛋白NusA-hRI的高效异源表达、纯化及活性分析[J]. 中国生物工程杂志, 2019, 39(3): 21-28.

Da-wei FU,Ying-ying SUN,wei XU. Efficient Heterologous Expression, Purification and Activity Analysis of Fusion Protein NusA-hRI. China Biotechnology, 2019, 39(3): 21-28.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190304 或 https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I3/21

表1 正交试验因素水平表

图1 PCR 扩增 hRI 基因

图2 酶切检测重组表达载体

图3 菌落 PCR 鉴定重组表达载体

图4 hRI诱导表达的12%聚丙烯酰胺凝胶分析

图5 hRI 可溶性表达的 12% 聚丙烯酰胺凝胶分析

表2 培养条件单因素实验设计及结果

表3 L9 (34)正交设计表及结果分析

表4 正交试验方差分析表

图6 磁珠纯化 hRI 的 12% 聚丙烯酰胺凝胶分析

图7 RNase/Sepharose 亲和层析纯化

图8 hRI酶活性检测

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