title: Expression and purification of truncated diphtheria toxin, DT386, inEscherichia coli: An attempt for production of a new vaccine against diphtheria.
abstract: The aim of this study was to produce a recombinant protein consisting of the catalytic and translocation domains of diphtheria toxin for its later application as a vaccine candidate againstCorynebacterium diphtheria. To achieve this goal, at first, the amino acid sequence of DT386 was used for prediction of T and B cell epitopes using on-line servers. The DT386 coding sequence was synthesized and subcloned into theNcoIandXhoIsites of pET28a plasmid and recombinant pET28a plasmid was used to transformEscherichia coliBL21 (DE3) host cells. Afterwards, recombinant cells were selected and subjected to induction of expression by 1 mM isopropyl β-D-1-thiogalactopyranoside, (IPTG). Expression of the desired protein was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, and finally, the recombinant protein was purified using nickel affinity chromatography. The results of epitope prediction using on-line servers established the ability of DT386 for stimulation of immune system against diphtheria toxin. Restriction digestion of the recombinant plasmids usingNcoIandXhoIenzymes confirmed the fidelity of cloning by producing a band of about 1200 bp. SDS-PAGE analysis following induction of expression and also purification step confirmed the expression of the desired protein by showing a band of about 45 kDa. In addition, Western blot analysis using anti-6X-His antibody confirmed the identity of the expected protein. In conclusion, in the present study we amplified and cloned the coding sequence of DT386 fragment, followed by its expression byE. coliBL21 (DE3) cells. Then, the expressed protein was purified and will be used for later studies of evaluation of its immunogenic properties.
authors.affiliation: Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, I.R. Iran.
authors.forename: Fatemeh
authors.initials: F
authors.lastname: Shafiee
authors.affiliation: Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, I.R. Iran.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Rabbani
authors.affiliation: Biotechnology Research Center, Venom and Biotherapeutics Molecules Laboratory, Pasteur Institute of Iran, Tehran, I.R. Iran.
authors.forename: Mahdi
authors.initials: M
authors.lastname: Behdani
authors.affiliation: Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, I.R. Iran.
authors.forename: Ali
authors.initials: A
authors.lastname: Jahanian-Najafabadi
keywords: DT386; Diphtheria toxin; Immunoinformatics; Vaccine candidate