Expression and Purification of Recombinant Catalytic Domain of ‎Botulinum Neurotoxin Type E from a Synthetic Gene

Aims: Botulinum neurotoxins are the strongest known bacterial toxins that cause muscle paralysis due to inhibition of acetylcholine release. Design of inhibitors is still pursued as a major strategy for intracellular inhibition of poisoning caused by these toxins. Investigation of the potential function of design inhibitors, pure poison or catalytic area is essential. The aims of present study were expression and purification of recombinant catalytic domain of botulinum neurotoxin type E (BoNT/E) Type E from a synthetic gene. Materials and Methods: In this experimental study, the sequence of the botulinum neurotoxin type E light chain was adopted from GeneBank and codons were optimized according to E.coli BL۲۱ (DE۳) codon usage. Other bioinformatics tools were exploited to reach the optimum expression of the gene in the mentioned host. The resultant (gene) was then ordered to synthesize and cloning in pET۲۸a (+) expression vector. The recombinant vector was transferred into E. coli BL۲۱ (DE۳) host cells. The expression of the protein was induced by addition of IPTG. The expression conditions were changed to obtain a soluble expression of the protein. Then, the protein was purified by an affinity chromatography, followed by a further purification with amicon filter. SDS-PAGE was used to evaluate expression and purification of the protein and Western blotting was performed to confirm the expressed protein. Finding: Codon Adaptation Index of the gene increased to ۰.۸۵. The third predicted structure showed good quality. The thermodynamic analysis of the mRNA structure showed that the predicted structure is stable. The soluble expression was obtained in ۱۸°C and ۱۸h induction by ۱ mM IPTG. Protein production with higher more than ۹۰% purity was confirmed. Conclusion: Optimization of the protein expression conditions resulted in producing the solution in the culture medium by E. coli BL۲۱ as host.

کلیدواژه ها:

Botulinum toxin Type –E ، Immunoglobulin Light Chain ، Catalytic Domain ، ‎Recombinant Proteins ، توکسین بوتولینوم تیپ E ، زنجیره سبک ، ناحیه کاتالیتیک ، پروتئین های نوترکیب