Cloning and optimization of expression and purification of the catalytic domain of adenylate cyclase toxin from an Iranian strain of Bordetella pertussis

Adenylate cyclase toxin is one of the immunogenic virulence factors secreted by Bordetella pertussis, that consists of catalytic domain corresponding to the 400 N-terminal residues called AC. This domain can stimulate cAMP synthesis in eukaryotic cells, and it has an important role in development of pertussis. It also can be used as a cell biology tool and in anti-cancer drugs. The aim of this study was to achieve high expression of the antigenic properties of the AC to evaluate its use in vaccines, adjuvant or the provision of diagnostic kits.
In this experimental study, AC domain amplified by PCR reaction, and cloned into pET28a vector and expressed in the E. coli BL21 (DE3) host. After the optimization of expression, protein analysis by SDS PAGE gel and Western blot analysis were performed. Purification was carried out on column chromatography (NI-NTA).
AC fragment was cloned successfully into a pET28a vector and under expression system with a concentration of 0.5 mM IPTG induction after 4 hours at 37 °C, AC can be expressed as a function of maximum yields 25mg/L and 95% purity.
This study showed that the induction of expression using only 0.5 mM IPTG can result in the over expression of the catalytic domain of protein ACT with maximum purity. This amount is appropriate for many applications, including research and diagnosis.