1- Tehran
Abstract: (11027 Views)
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.
Article Type:
Research Paper |
Subject:
Biotechnology Received: 2011/10/17 | Accepted: 2012/09/10 | Published: 2013/03/10