EXPRESSION AND CHARACTERIZATION OF RECOMBINANT HUMAN EOSINOPHIL-DERIVED NEUROTOXIN AND EOSINOPHIL-DERIVED NEUROTOXIN-ANTI-TRANSFERRIN RECEPTOR SFV

The gene for the human recombinant eosinophil-derived neurotoxin (rEDN ) was synthesized and fused to the gene encoding a single chain antibo dy (sFv) to the human transferrin receptor (EDNsFv). Both rEDN and EDN sFv were expressed as insoluble proteins in inclusion bodies in Escher ichia coli BL21(DE3). Following denaturation and renaturation, EDN and EDNsFv were partially purified by chromatography on heparin-Sepharose . Final purification of EDN was achieved by Sephadex G-100, whereas ED NsFv which contained a g-histidyl residue carboxyl terminus was highly purified using the metal chelate resin, Ni2+-nitriloacetic acid. Wher eas the recombinant EDN had ribonuclease activity that was similar to the native protein, the fusion protein had enzymatic activity that was 6-13% that of native EDN. The fusion protein was able to bind to the human transferrin receptor. In contrast to rEDN that had no inherent c ytotoxicity to human tumor cells, the EDNsFv fusion protein was cytoto xic to human leukemia cells that express the human transferrin recepto r with an IC50, 0.2-1 nM. At 1.3 nM EDNsFv, no cytotoxicity was observ ed on cells that lack the human transferrin receptor. Free antibody to the human transferrin receptor, E6, inhibited the cytotoxicity of the EDNsFv. Human enzymes may be engineered to acquire cytotoxic properti es by fusing them to antibodies. Thus, they may be candidates for the construction of immunofusion proteins that may be less immunogenic tha n immunotoxins containing bacterial- or plant-derived toxin moieties.