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

Introduction Pseudomonas aeruginosa exotoxin A (PE) is one of the most potent toxins capable of modifying specific target proteins in mammalian cells. PE is consisted of three functional and structural domains as shown by x-ray crystallography. Domain Ia (amino acids 1-252) is responsible for cell recognition, domain II (amino acids 253-364) is involved in the translocation of the toxin to the cytosol and domain III (amino acids 400-613) catalyzes the irreversible ADP-ribosylation of elongation factor 2 (EF-2), leading to inhibition of protein synthesis and consequently to cell death. The exact function of domain Ib (amino acids 345-404) has not been defined. In recent years, studies on the molecular mechanism of the PE cytotoxicity showed an induction of apoptosis by activation of caspase-3 and caspase-8. It has shown that high cytotoxicity of PE can be exploited for construction of immunotoxins, a new class of immunotherapeutic agents against cancer. In these molecules, the active enzymatic domain of PE is specifically directed to tumor specific antigens. For immunotoxin design, a truncated form of PE, lacking the receptor binding domain, is conjugated to a ligand which is specific to an antigen on the tumor cell surface. The toxin domain internalizes to the cells cytosol and catalytically kills tumor cells. RFB4 (dsFv)-PE38 is an example for a disulfide-stablized recombinant immunotoxin, also known as BL22, that is constructed by fusion of Fv domain of anti-CD22 monoclonal antibody RFB4 to PE38 (Li 1989, Pastan 2007). It shows specific cytotoxicity in patients with a variety of B-cell malignancies. The objective of the present study was to clone and express the truncated PE in E. coli for the production of recombinant toxin.