title: Construction, expression and characterization of chimaeric toxins containing the ribonucleolytic toxin restrictocin: intracellular mechanism of action.
abstract: Restrictocin is a ribonucleolytic toxin produced by the fungus Aspergillus restrictus. 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 Escherichia coli, 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 in vitro translation 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.
authors.affiliation: Immunochemistry Laboratory, National Institute of Immunology, Aruna Asaf Ali Road, New Delhi-110067, India.
authors.forename: D
authors.initials: D
authors.lastname: Rathore
authors.forename: J K
authors.initials: JK
authors.lastname: Batra
mesh_terms: D000485:Allergens; D052179:Antigens, Plant; D003001:Cloning, Molecular; D003603:Cytotoxins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D004347:Drug Interactions; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D006801:Humans; D007700:Kinetics; D011480:Protease Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D011500:Protein Synthesis Inhibitors / Q000633:toxicity; D011990:Receptors, Transferrin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D012260:Ribonucleases / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D014407:Tumor Cells, Cultured
chemical_list: C081947:ASPF1 protein, Aspergillus fumigatus; D000485:Allergens; D052179:Antigens, Plant; D003603:Cytotoxins; D005656:Fungal Proteins; D011480:Protease Inhibitors; D011500:Protein Synthesis Inhibitors; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; C036196:MITF protein, Aspergillus restrictus; D012260:Ribonucleases