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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2012, Vol. 32 Issue (08): 62-67    
技术与方法     
无标签重组人硫氧还蛋白的大规模表达、纯化及活性检测
郭云萍1, 孙璐1, 张立剑1, 王增禄2, 高超1, 杨强1, 刘毅1, 张英起2, 屈延1, 陶凌1
1. 第四军医大学西京医院 西安 710032;
2. 第四军医大学药学系生物技术中心 西安 710032
Expression, Purification and Characterization of Non-taged Recombinant Human Thioredoxin
GUO Yun-ping1, SUN Lu1, ZHANG Li-jian1, WANG Zeng-lu2, GAO Chao1, YANG Qiang1, LIU Yi1, ZHANG Ying-qi2, QU Yan1, TAO Ling 1
1. Department of Cardiology, XiJing Hospital, The Fourth Military Medical University, Xi’an 710032, China;
2. Biotechnology Center, School of Pharmacy, The Fourth Military Medical University, Xi’an 710032, China
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摘要: 目的:利用基因工程的方法原核表达无标签的重组人硫氧还蛋白(rhTrx)并对其进行大规模表达、纯化和鉴定。方法:从人胚胎肾HEK293细胞中提取总RNA,反转录合成cDNA,经PCR扩增、酶切后连入pET-22b(+)载体构建重组质粒,重组质粒转化大肠杆菌BL21(DE3)感受态细胞,IPTG诱导表达,经两步离子交换层析纯化重组蛋白,采用SDS-PAGE、Western blotting、HPLC、MALDI-TOF-MS及经典的胰岛素二硫键还原法对重组蛋白进行鉴定。结果:构建成功了rhTrx基因表达载体;实现了rhTrx在原核细胞中的可溶性表达;纯化出的蛋白经SDS-PAGE和Western blotting分析证实为rhTrx;HPLC和MALDI-TOF-MS分析表明,纯化出的目的蛋白纯度大于95%;胰岛素二硫键还原法证实纯化出的 rhTrx具有生物学活性。结论:成功构建了rhTrx的原核表达体系,建立了rhTrx的纯化和鉴定方法,为其进一步的理论研究和生产开发提供了有效基础数据。
关键词: 重组人硫氧还蛋白(rhTrx)表达纯化生物活性    
Abstract: Objective:To construct prokaryotic expression system for mass production of recombinant human thioredoxin and establish the purification process of thioredoxin. Methods: Total RNA was extracted from HEK293 (human embryonic kidney cells). The thioredoxin coding sequence was subcloned into the pET-22b(+) vector after amplified by PCR.The recombinant plasmids were transformed into E.coli BL21(DE3), and the thioredoxin was expressed with IPTG induction. The expressed thioredoxin was purified by two-step ion exchange chromatography and tested by SDS-PAGE, Western blotting, MALDI-TOF-M, HPLC, and insulin disulfide reduction assay for identification, purity assay and activity determination, respectively. Results: Gene sequencing demonstrated that thioredoxin coding sequence was cloned into pET-22b(+) vector successfully. The prokaryotic expression system achieved high yield of thioredoxin (180 mg/5L of fermentation broth), which was identified by Western blotting and MALDI-TOF-MS, with an estimated the molecular weight of 12 000. The purity of thioredoxin is more than 95%. The activity of purified thioredoxin had the same activity as the standard control. Conclusion: The prokaryotic expression system could achieve mass production of recombinant human thioredoxin, which can be highly purified by two-steps ion exchange chromatography. This preliminary study provides the foundation for the large-scale industrial production of thioredoxin.
Key words: Recombinant human thioredoxin    Expression and purification    Biological activity
收稿日期: 2012-03-23 出版日期: 2012-08-25
ZTFLH:  Q786  
基金资助: 国家科技重大专项(2009ZXJ09004-088)、国家自然科学基金(81070676)、国家重大新药创制计划(2012ZX09J12108-06B)资助项目
通讯作者: 陶凌     E-mail: lingtao2006@gmail.com
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引用本文:

郭云萍, 孙璐, 张立剑, 王增禄, 高超, 杨强, 刘毅, 张英起, 屈延, 陶凌. 无标签重组人硫氧还蛋白的大规模表达、纯化及活性检测[J]. 中国生物工程杂志, 2012, 32(08): 62-67.

GUO Yun-ping, SUN Lu, ZHANG Li-jian, WANG Zeng-lu, GAO Chao, YANG Qiang, LIU Yi, ZHANG Ying-qi, QU Yan, TAO Ling. Expression, Purification and Characterization of Non-taged Recombinant Human Thioredoxin. China Biotechnology, 2012, 32(08): 62-67.

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https://manu60.magtech.com.cn/biotech/CN/        https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I08/62

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