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| 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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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.
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Received: 23 March 2012
Published: 25 August 2012
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Cite this article:
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.
URL:
https://manu60.magtech.com.cn/biotech/ OR https://manu60.magtech.com.cn/biotech/Y2012/V32/I08/62
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