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

Abstract

Background: Tetanus is an infectious disease caused by clostridium tetani 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.

Heterologous expression, molecular studies and biochemical characterization of a novel alkaline esterase gene fromBacillus thuringiensisfor detergent industry

“…This technique has been successfully employed for the purication of different recombinant proteins for various purposes. [53][54][55][56][57] The molecular mass of puried recombinant esterase enzyme was 29 kDa as analyzed by SDS-PAGE (Fig. 3).…”

Tetanus Toxin Fragment C: Structure, Drug Discovery Research and Production

“…Purification [104,105,107,108,110,152] and characterization [108,110,122,133,152] steps are then usually the last ones performed in protein production to finally obtain the required quality. In 2020, Chai et al established a simple method to purify TTFC by ion-exchange chromatography [153].…”