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

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 toxin. The genomic DNA extracted from P. aeruginosa PAO1 was used in a polymerase chain reaction (PCR) technique containing the primers for amplification of domain 2-3 of PE gene. The PCR product was then cloned into the pET-22b vector and transformed into E. coli BL21 cells. The expression of soluble truncated PE was optimized in the presence of different additives and the His-tagged recombinant PE38 was purified by Nickel affinity chromatography. Sequencing of the cloned fragment confirmed the identity of the fragment as PE domain 2-3. Analyzing the recombinant protein expression and purification by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting showed that the present expression system is efficient for the production of recombinant truncated exotoxin A. This recombinant protein can be used for the construction of toxin conjugates against different cancers.