title: Immunotoxin construction with a ribosome-inactivating protein from barley.
abstract: The aim of this study was to determine the suitability of a ribosome-inactivating protein (RIP) from barley endosperm for use as an immunotoxin. This barley RIP is identical with the 30-kDa protein first reported by Coleman and Roberts [(1982) Biochim. Biophys. Acta 696, 239] and sequenced by Asano and co-workers [(1986) Carlsberg Res. Commun. 51, 75]. Use of the terms barley toxin I, II, and III is proposed to describe the three isoforms resolved by cation-exchange chromatography. An improved procedure for isolating the protein involving the steps of aqueous extraction, ammonium sulfate precipitation, and cation-exchange HPLC is described. Barley toxin II retained activity after exposure to ca. 40% acetonitrile and 0.1% trifluoroacetic acid or lyophilization. In a comparative study using the rabbit reticulocyte lysate assay, the protein was about 68% and 30% as potent as gelonin and ricin A-chain (RTA), respectively. Introduction of SH groups with 2-iminothiolane resulted in a substantial loss of activity as the number of thiol groups approached four. Therefore, it was necessary to limit thiolation to an average of one to two SH groups per toxin molecule. Anti-transferrin receptor-based immunotoxins constructed with RTA, gelonin, and barley toxin II exhibited comparable cytotoxicity against a human colon tumor cell line. We conclude that the availability of raw material, ease of purification, and stability of barley toxin II to lyophilization and denaturing conditions render it a suitable protein for the construction of immunotoxins.
authors.affiliation: Akzo Pharma/Organon Teknika Corporation, Biotechnology Research Institute, Rockville, Maryland 20850-4373.
authors.forename: R F
authors.initials: RF
authors.lastname: Ebert
authors.forename: L A
authors.initials: LA
authors.lastname: Spryn
mesh_terms: D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000627:therapeutic use*; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid; D003110:Colonic Neoplasms / Q000188:drug therapy; D004591:Electrophoresis, Polyacrylamide Gel; D001467:Hordeum; D007096:Imidoesters; D000922:Immunotoxins / Q000627:therapeutic use; D008970:Molecular Weight; D009699:N-Glycosyl Hydrolases; D037121:Plant Lectins; D010940:Plant Proteins / Q000008:administration & dosage / Q000302:isolation & purification / Q000627:therapeutic use*; D011990:Receptors, Transferrin / Q000276:immunology*; D054789:Ribosome Inactivating Proteins, Type 1; D012276:Ricin / Q000627:therapeutic use; D014118:Toxins, Biological; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D000970:Antineoplastic Agents; D007096:Imidoesters; D000922:Immunotoxins; D037121:Plant Lectins; D010940:Plant Proteins; D011990:Receptors, Transferrin; D054789:Ribosome Inactivating Proteins, Type 1; D014118:Toxins, Biological; C005905:methyl 4-mercaptobutyrimidate; C025674:GEL protein, Gelonium multiflorum; D012276:Ricin; D009699:N-Glycosyl Hydrolases; C487943:ribosome-inactivating protein, Hordeum vulgare