Rare Codons Optimizer Host Strain of E. coli Improves Expression of Clostridium septicum Alpha Toxin Gene

Abstract

Aims: Clostridium septicum is an anaerobic, gram positive bacterium that is able to form resistant spores. It produces numerous toxins that cause many diseases such as gas gangrene in humans and braxy in farm animals. In the present study, an attempt was done to express an active fragment of alpha toxin gene of C. septicum vaccine strain, in E. coli strains BL21 (DE3) and Rosetta (DE3) strains. Methodology: In the present study, the active fragment of alpha toxin gene of C. septicum vaccine strain was amplified and ligated with pJET1.2blunt cloning vector and after cloning was extracted from the pJETαsep recombinant cloning vector. After purification and evaluation, it was ligated with pET22b(+) expression vector and the pET22αsep was transformed into E. coli strains BL21(DE3) and Rosetta(DE3) strains. Results: Results showed that a recombinant protein was expressed as a soluble protein after IPTG induction in Rosetta (DE3) rare codons optimizer host strain, but not in BL21 (DE3). Further optimization on expression conditions of the recombinant alpha toxin protein was achieved by incubating the culture of recombinant E. coli/ Rosetta (DE3[pET22αsep] cells in 37ºC and induction with 0.5, 1.0 and 1.5 mM IPTG for 3-6 h. Protein expression was evaluated by SDS-PAGE and the recombinant alpha toxin protein was purified using Ni-NTA resin and for its reconfirmation was analysed by Western blot. Conclusions: We concluded that, E. coli strain Rosetta (DE3) is a suitable expression host for production of C. septicum alpha toxin and the obtained recombinant plasmid could be used for further research on production of recombinant vaccine against braxy disease.