title: Expression and stability of the nontoxic component of the botulinum toxin complex.
abstract: Clostridium botulinum produces botulinum neurotoxin (BoNT) as a large toxin complex associated with nontoxic-nonhemagglutinin (NTNHA) and/or hemagglutinin components. In the present study, high-level expression of full-length (1197 amino acids) rNTNHA from C. botulinum serotype D strain 4947 (D-4947) was achieved in an Escherichia coli system. Spontaneous nicking of the rNTNHA at a specific site was observed during long-term incubation in the presence of protease inhibitors; this was also observed in natural NTNHA. The rNTNHA assembled with isolated D-4947 BoNT with molar ratio 1:1 to form a toxin complex. The reconstituted toxin complex exhibited dramatic resistance to proteolysis by pepsin or trypsin at high concentrations, despite the fact that the isolated BoNT and rNTNHA proteins were both easily degraded. We provide definitive evidence that NTNHA plays a crucial role in protecting BoNT, which is an oral toxin, from digestion by proteases common in the stomach and intestine.
authors.affiliation: Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture, Abashiri, Japan.
authors.forename: Keita
authors.initials: K
authors.lastname: Miyata
authors.forename: Tohru
authors.initials: T
authors.lastname: Yoneyama
authors.forename: Tomonori
authors.initials: T
authors.lastname: Suzuki
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Kouguchi
authors.forename: Ken
authors.initials: K
authors.lastname: Inui
authors.forename: Koichi
authors.initials: K
authors.lastname: Niwa
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Watanabe
authors.forename: Tohru
authors.initials: T
authors.lastname: Ohyama
mesh_terms: D000595:Amino Acid Sequence; D001905:Botulinum Toxins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D003014:Clostridium botulinum; D004926:Escherichia coli / Q000235:genetics; D010434:Pepsin A / Q000737:chemistry; D055550:Protein Stability; D014357:Trypsin / Q000737:chemistry
chemical_list: D014357:Trypsin; D010434:Pepsin A; D001905:Botulinum Toxins