The Protein Journal

, Volume 32, Issue 4, pp 288–296

Purification and Characterization of Neurotoxin Complex from a Dual Toxin Gene Containing Clostridium Botulinum Strain PS-5

  • Ajay K. Singh
  • Amita Sachdeva
  • Jeffrey A. DeGrasse
  • Timothy R. Croley
  • Larry H. Stanker
  • David Hodge
  • Shashi K. Sharma


Botulinum neurotoxins are produced as a toxin complex (TC) which consists of neurotoxin (NT) and neurotoxin associated proteins. The characterization of NT in its native state is an essential step for developing diagnostics and therapeutic countermeasures against botulism. The presence of NT genes was validated by PCR amplification of toxin specific fragments from genomic DNA of Clostridium botulinum strain PS-5 which indicated the presence of both serotype A and B genes on PS-5 genome. Further, TC was purified and characterized by Western blotting, Digoxin-enzyme linked immunosorbent assay, endopeptidase activity assay, and Liquid chromatography–Mass spectrometry. The data showed the presence of serotype A specific neurotoxin. Based on the analysis of neurotoxin genes and characterization of TC, PS-5 strain appears as a serotype A (B) strain of C. botulinum which produces only serotype A specific TC in the cell culture medium.


Botulism Neurotoxin complex Neurotoxin associated proteins PCR ELISA Western blot Endopeptidase activity 



Botulinum neurotoxin


Toxin complex


Neurotoxin associated proteins


Nontoxic non-hemagglutinin




Genomic DNA


Digoxigenin-enzyme-linked immunosorbent assay


Liquid chromatography-Mass spectrometry


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Copyright information

© Springer Science+Business Media New York (outside the USA)  2013

Authors and Affiliations

  • Ajay K. Singh
    • 1
  • Amita Sachdeva
    • 1
  • Jeffrey A. DeGrasse
    • 2
  • Timothy R. Croley
    • 2
  • Larry H. Stanker
    • 3
  • David Hodge
    • 4
  • Shashi K. Sharma
    • 1
  1. 1. Divisions of Microbiology, Center for Food Safety and Applied NutritionFood and Drug AdministrationCollege ParkUSA
  2. 2.Analytical Chemistry, Center for Food Safety and Applied NutritionFood and Drug AdministrationCollege ParkUSA
  3. 3.Western Regional Research Center, Agricultural Research ServiceUnited States Department of AgricultureAlbanyUSA
  4. 4.Department of Homeland SecurityWashingtonUSA

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