title: Purification and characterization of nontoxic protein complex from serotype D 4947 botulinum toxin complex.
abstract: The large-sized botulinum toxin complex (L-TC) is composed of botulinum neurotoxin (BoNT) and nontoxic proteins, e.g. nontoxic nonhemagglutinin (NTNHA) and three types of hemagglutinins (HAs; HA-33, HA-17 and HA-70). The nontoxic proteins play a critical role in L-TC oral toxicity by protecting the BoNT in the digestive tract, and facilitating absorption of the L-TC across the intestinal wall. Under alkaline conditions, the L-TC separates into BoNT and the nontoxic protein complex (NC). In this study, we established a two-step procedure to yield highly pure NC from the L-TC produced by Clostridium botulinum serotype D strain 4947 in which the NC was isolated from the L-TC by gel filtration under alkaline conditions followed by immunoprecipitation with an anti-BoNT antibody to remove contaminating BoNT from the NC fraction. Western blotting and electrophoretic analysis showed that the highly purified NC fraction had only very slight or no BoNT contamination. In addition, the purified NC fraction showed no intraperitoneal (ip) toxicity to mice at a dose of 38 ng per animal whereas the L-TC exhibited an ip median lethal dose of 0.38 ng per mouse. The highly purified NC displayed the same hemagglutination titer as the L-TC. The NC, as well as the L-TC, demonstrated cell binding and monolayer transport in the rat intestinal epithelial cell line IEC-6. Consequently, the highly purified NC can function as a "delivery vehicle" even without the BoNT.
authors.affiliation: Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri, 099-2493, Japan.
authors.forename: Keita
authors.initials: K
authors.lastname: Miyata
authors.forename: Yoshimasa
authors.initials: Y
authors.lastname: Sagane
authors.forename: Ken
authors.initials: K
authors.lastname: Inui
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Miyashita
authors.forename: Tomonori
authors.initials: T
authors.lastname: Suzuki
authors.forename: Keiji
authors.initials: K
authors.lastname: Oguma
authors.forename: Tohru
authors.initials: T
authors.lastname: Ohyama
authors.forename: Koichi
authors.initials: K
authors.lastname: Niwa
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Watanabe
mesh_terms: D000818:Animals; D001905:Botulinum Toxins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000633:toxicity*; D001906:Botulism / Q000382:microbiology; D002460:Cell Line; D002850:Chromatography, Gel; D047969:Clostridium botulinum type D / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006384:Hemagglutination / Q000187:drug effects; D006386:Hemagglutination Tests; D006736:Horses; D047468:Immunoprecipitation; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats
chemical_list: C096325:botulinum toxin type D; D001905:Botulinum Toxins