Expression and stability of the nontoxic component of the botulinum toxin complex.
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.
Miyata
Keita
K
Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture, Abashiri, Japan.
Yoneyama
Tohru
T
Suzuki
Tomonori
T
Kouguchi
Hirokazu
H
Inui
Ken
K
Niwa
Koichi
K
Watanabe
Toshihiro
T
Ohyama
Tohru
T
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
D014357:Trypsin; D010434:Pepsin A; D001905:Botulinum Toxins