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Expression and Purification of Tetanus Toxin Fragment C in Escherichia coli BL21(DE3)

[ Vol. 27 , Issue. 11 ]

Author(s):

Pengdi Chai, Xiuying Pu, Jianqiang Li*, Xiaoyu Xia, Jun Ge, Amiao Luo, Hui Su, Weijie Zhang and Jianzhong Ma   Pages 1132 - 1140 ( 9 )

Abstract:


Background: Tetanus is an infectious disease caused by Clostridium secreting tetanus toxin in anaerobic environment. The fragment C of Tetanus toxin (TTc) has been widely studied as a candidate vaccine to replace the existing tetanus toxoid vaccine.

Objective: In this study, we established a simple method to purify recombinant protein TTc with ion-exchange chromatography from Escherichia coli expression systems.

Methods: The TTc gene sequence was cloned into pET26b (+) vector and transferred to E. coli BL21 (DE3) for expression. The fermentation conditions (IPTG concentration, Induction temperature, Induction time) were optimized to obtain more soluble proteins. The soluble proteins were purified by Anion exchange chromatography and Cation exchange chromatography. The sequence of columns in the purification process was discussed. Finally, the stability of purified TTc protein were determined, the secondary structure of the purified TTc protein was determined by circular dichroism. The molecular weight of the purified TTc protein was determined by liquid chromatograph- mass spectrometer. Furthermore, we verified the immunogenicity of the purified protein in mice.

Results: The purity of TTc improved from 34% to 88% after the first anion exchange column, and the final yield of recombinant TTc (purity > 95%) can reach 84.79% after the following cation exchange chromatography. The recombinant TTc had a molecular weight of 51.737 KDa, was stable at 4 °C and weak alkaline environment, was a β-sheet secondary structure, and had strong immunogenicity.

Conclusion: The purification method we developed might be an efficient method for the industrial production of tetanus recombinant TTc vaccine.

Keywords:

Tetanus, the fragment C, tetanus toxin, purification, molecular weight, immunogenicity.

Affiliation:

School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, Jiangsu Theravac Bio-Pharmaceutical Co., Ltd, Nanjing 210042, School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, Jiangsu Theravac Bio-Pharmaceutical Co., Ltd, Nanjing 210042, School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050

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