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

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Abstract

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.

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Abbreviations

BoNT:

Botulinum neurotoxin

TC:

Toxin complex

NAPs:

Neurotoxin associated proteins

NTNH:

Nontoxic non-hemagglutinin

HAs:

Hemagglutinins

gDNA:

Genomic DNA

DIG-ELISA:

Digoxigenin-enzyme-linked immunosorbent assay

LC-MS:

Liquid chromatography-Mass spectrometry

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Acknowledgments

Authors thank Dr. Eric W. Brown for valuable discussions and advice. This work was supported by the Department of Homeland Security through Inter Agency Grant. The views or opinions presented in this article are those of the authors and should not be construed as an official view of the U.S. Food and Drug Administration.

Conflict of interest

Authors declare that there are no conflicts of interest.

Ethical standard

The experiments comply with the current laws of the United States of America for ethical standards.

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Authors and Affiliations

  1. Divisions of Microbiology, Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, MD, 20740, USA

    Ajay K. Singh, Amita Sachdeva & Shashi K. Sharma

  2. Analytical Chemistry, Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, MD, 20740, USA

    Jeffrey A. DeGrasse & Timothy R. Croley

  3. Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, CA, 94170, USA

    Larry H. Stanker

  4. Department of Homeland Security, Washington, DC, 20528, USA

    David Hodge

Authors
  1. Ajay K. Singh
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  2. Amita Sachdeva
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  3. Jeffrey A. DeGrasse
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  4. Timothy R. Croley
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  5. Larry H. Stanker
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  6. David Hodge
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  7. Shashi K. Sharma
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Correspondence to Ajay K. Singh or Shashi K. Sharma.

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Singh, A.K., Sachdeva, A., DeGrasse, J.A. et al. Purification and Characterization of Neurotoxin Complex from a Dual Toxin Gene Containing Clostridium Botulinum Strain PS-5. Protein J 32, 288–296 (2013). https://doi.org/10.1007/s10930-013-9486-1

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  • DOI: https://doi.org/10.1007/s10930-013-9486-1

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