Optimization of expression and in vitro characteristics evaluation of Pseudomonas aeruginosa recombinant exotoxin A (domains I and II)

P. aeruginosa exotoxin A plays an important role in virulence of the bacterium and its neutralization can abolish the P. aeruginosa pathogenicity. Therefore, we showed optimization of recombinant production and in vitro antigenic characteristics evaluation of exotoxin A (domains I and II) as a vaccine candidate. In this study, after DNA extraction and amplification with PCR, gene fragment was ligated to Pet22b vector and transferred to E.coli BL21. The protein expression was evaluated by SDS-PAGE method. The Ni-NTA affinity chromatography was used for recombinant protein purification. Then, the reactivity of recombinant exotoxin A with anti-exotoxin A antibody (Sigma) and sera from P. aeruginosa infected patients was evaluated by western blotting method. Sequencing of cloned gene showed that the sequence of exoA I-II gene was in accordance with exoA I-II from P. aeruginosa PAO1. SDS-PAGE analysis indicated that expression of recombinant protein with a molecular weight of 45kD. The results showed, 4 hours induction with IPTG = 0.5mM/m in 28˚C is optimum condition for expression protein. Western blot analysis of the purified protein demonstrated that ExoA I-II could be recognized by antibody against native exotoxin A and the serum of patients with P. aeruginosa infection. These results suggest that recombinant exoA I-II protein can be produced in the laboratory and this system can be used for large scale production of this protein for subsequent immunological studies.