title: Use of blue-sepharose for purification of immunotoxin containing type 1 ribosome-inactivating protein, gelonin.
abstract: This paper describes a method suitable for purifying immunotoxin containing type 1 ribosome-inactivating protein, gelonin. The separation of free (unreacted) 80G, a monoclonal antibody against alpha-fetoprotein (AFP), from semipurified 80G-gelonin conjugate was unsuccessful by conventional CM-Sepharose ion-exchange chromatography because the isoelectric point of the conjugate did not increase enough to reach that of gelonin alone. In contrast, Blue Sepharose affinity chromatography could efficiently separate free 80G from the semipurified conjugate because the conjugate was bound to the column by its gelonin moiety while free 80G was not in buffer containing NaCl of a particular concentration range. However, a small amount of conjugate containing gelonin modified with N-succinimidyl 3-(2-pyridyldithio)propionate, but not with 2-iminothiolane, could not bind to the column. The conjugate purified by the use of Blue Sepharose showed selective cytotoxicity against AFP-producing human hepatoma cells.
authors.affiliation: Shionogi Research Laboratories, Shionogi & Co., Ltd., Osaka, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Masuda
authors.forename: K
authors.initials: K
authors.lastname: Hirano
authors.forename: Y
authors.initials: Y
authors.lastname: Takagishi
mesh_terms: D000911:Antibodies, Monoclonal; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology; D002847:Chromatography, Agarose / Q000379:methods*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D000922:Immunotoxins / Q000032:analysis*; D007526:Isoelectric Point; D010940:Plant Proteins / Q000032:analysis / Q000302:isolation & purification*; D011500:Protein Synthesis Inhibitors / Q000032:analysis / Q000302:isolation & purification*; D054789:Ribosome Inactivating Proteins, Type 1; D012685:Sepharose / Q000031:analogs & derivatives*; D014407:Tumor Cells, Cultured; D000509:alpha-Fetoproteins / Q000276:immunology / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000922:Immunotoxins; D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D054789:Ribosome Inactivating Proteins, Type 1; D000509:alpha-Fetoproteins; C029483:Blue-Sepharose; C025674:GEL protein, Gelonium multiflorum; D012685:Sepharose