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Showing 2 results for Light Chain
Firouz Ebrahimi, , , , , ,
Volume 3, Issue 2 (11-2012)
Abstract
Botulinum neurotoxins are the most known toxic biological compounds, which cause neuroparalysis. The enzymatic activity of these enzymes causes the inhibition of acetylcholine release. The aim of this study is the recombinant production and high purification of BoNT/A light chain and evaluation of its enzymatic activity. The sequence of this target gene was obtained from NCBI. After codon usage optimization for E. coli, the final gene sequence was ordered for the synthesis on pET28a (+). The recombinant expression vector was transformed into host cell E.coli BL21 (DE3). The expression process was performed under standard conditions. In order to the protein production in a soluble form, optimization of host cell culturing and protein expression was carried out. The expressed protein was purified by Ni-NTA affinity chromatography, and confirmed by specific antibody.In this study, the high yield expression in soluble form was obtained at OD = 0.5, in 0.5 mM IPTG at 18°C in 18 hours. Western blot and ELISA analyses confirmed the BoNT/A light chain.The results indicated that the light chain of BoNT/A was produced in soluble form, and the purification process was performed with high quality so that the final protein was acquired with 98% purity index.
Volume 21, Issue 2 (7-2018)
Abstract
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 BL21 (DE3) 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 pET28a (+) expression vector. The recombinant vector was transferred into E. coli BL21 (DE3) 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 0.85. 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 18°C and 18h induction by 1 mM IPTG. Protein production with higher more than 90% purity was confirmed.
Conclusion: Optimization of the protein expression conditions resulted in producing the solution in the culture medium by E. coli BL21 as host.
