The Protein Journal

, Volume 31, Issue 5, pp 387–392 | Cite as

Purification and Characterization of Nontoxic Protein Complex from Serotype D 4947 Botulinum Toxin Complex

  • Keita Miyata
  • Yoshimasa Sagane
  • Ken Inui
  • Shin-Ichiro Miyashita
  • Tomonori Suzuki
  • Keiji Oguma
  • Tohru Ohyama
  • Koichi Niwa
  • Toshihiro Watanabe


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.


Clostridium botulinum Toxin complex Nontoxic protein complex Immunoprecipitation 



Botulinum neurotoxin


Nontoxic nonhemagglutinin




Toxin complex


Large-sized toxin complex


Middle-sized toxin complex


Nontoxic protein complex


Type D strain 4947


Horse radish peroxidase






ip median lethal dose


Sodium dodecyl sulfate–polyacrylamide gel electrophoresis


Coomassie brilliant blue


20 mM Tris-HCl buffer (pH 7.5), 150 mM NaCl and 0.1 % (v/v) Tween 20


Dulbecco’s modified Eagle medium


Fetal bovine serum


Light chain


Heavy chain


N-terminal heavy chain


C-terminal heavy chain



We thank Ms. Sayuri Kurihara, Mr. Takehiro Kawane, and Mr. Yuhma Yoshida for technical assistance. This research was supported by KAKENHI provided by MEXT/JSPS (for T. W., Grant No. 22590405).


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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Keita Miyata
    • 1
    • 2
  • Yoshimasa Sagane
    • 1
  • Ken Inui
    • 1
    • 2
  • Shin-Ichiro Miyashita
    • 1
  • Tomonori Suzuki
    • 3
  • Keiji Oguma
    • 3
  • Tohru Ohyama
    • 1
  • Koichi Niwa
    • 1
  • Toshihiro Watanabe
    • 1
  1. 1.Department of Food and Cosmetic Science, Faculty of BioindustryTokyo University of AgricultureAbashiriJapan
  2. 2.Japan Society for the Promotion of ScienceChiyoda-ku, TokyoJapan
  3. 3.Department of BacteriologyOkayama University Graduate School of Medicine, Dentistry and Pharmaceutical SciencesOkayamaJapan

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