Construction, expression and characterization of chimaeric toxins containing the ribonucleolytic toxin restrictocin: intracellular mechanism of action

摘要

pRestrictocin is a ribonucleolytic toxin produced by the fungus iAspergillus restrictus/i. Two chimaeric toxins containing restrictocin directed at the human transferrin receptor have been constructed. Anti-TFR(scFv)–restrictocin is encoded by a gene produced by fusing the DNA encoding a single-chain antigen-combining region (scFv) of a monoclonal antibody, directed at the human transferrin receptor, at the 5′ end of that encoding restrictocin. The other chimaeric toxin, restrictocin–anti-TFR(scFv), is encoded by a gene fusion containing the DNA encoding the single-chain antigen-combining region of antibody to human transferrin receptor at the 3′ end of the DNA encoding restrictocin. These gene fusions were expressed in iEscherichia coli/i, and fusion proteins purified from the inclusion bodies by simple chromatography techniques to near-homogeneity. The two chimaeric toxins were found to be equally active in inhibiting protein synthesis in a cell-free iin vitro/itranslation assay system. The chimaeric toxins were selectively toxic to the target cells in culture with potent cytotoxic activities. However, restrictocin–anti-TFR(scFv) was more active than anti-TFR(scFv)–restrictocin on all cell lines studied. By using protease and metabolic inhibitors, it can be shown that, to manifest their cytotoxic activity, the restrictocin-containing chimaeric toxins need to be proteolytically processed intracellularly and the free toxin or a fragment thereof thus generated is translocated to the target via a route involving the Golgi apparatus./p