Recombinant Expression and Purification of Pseudomonas aeruginosa Truncated Exotoxin A in Escherichia coli

Background: Pseudomonas (P.) aeruginosa exotoxin A (PE) is one of the most potent bacterial toxins ever been identified. It catalyzes ADP-ribosylation of cellular elongation factor 2 and specifically arrests protein synthesis leads to cell death. Different derivatives of PE have been used for construction of immunotoxins against cancers. The aim of this study was to clone and express a DNA fragment of PE encoding a truncated exotoxin lacking the cell binding domain of native exotoxin. Methods: The genomic DNA extracted from P. aeruginosa PAO1 was used in a polymerase chain reaction (PCR) containing the primers for amplification of domains 2-3 of PE gene. The PCR product was then cloned into the pET-22b vector and transformed into E. coli BL21 cells for expression. The His-tagged recombinant PE38 was purified by Nickel affinity chromatography and characterized. Results: Sequencing of the cloned fragment confirmed the identity of gene as PE domains 2-3. Analyzing the recombinant protein expression by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed high level expression of recombinant protein. Results of western blotting with anti native-exotoxin A showed proper conformational structure of purified recombinant protein. Conclusion: The results of this study indicated that the presented expression system is an efficient system for the production of recombinant truncated exotoxin A. This recombinant protein can be used for the construction of toxin conjugates against different cancers.