Cloning, Expression, Purification, and Characterization of<i> Clostridium botulinum</i> Neurotoxin Serotype F Domains

Authors

  • Santosh Dulal Department of Applied Molecular Biology, Sun Moon University, Asan-si, Chung-nam
  • Bhupal Ban Medy-Tox Inc., Cheongwon-gun, Chungbuk-do
  • Gi Hyoek Yang Medy-Tox Inc., Cheongwon-gun, Chungbuk-do
  • Hyun Ho Jung Department of Applied Molecular Biology, Sun Moon University, Asan-si, Chung-nam and Medy-Tox Inc., Cheongwon-gun, Chungbuk-do

DOI:

https://doi.org/10.3126/njb.v2i1.5634

Keywords:

Clostridium botulunium, Botulinum Neurotoxin Type F (BoNT/F) domains, cloning, recombinant protein expression, immunoreactivity

Abstract

The use of recombinant BoNT domains has been proposed as a means to develop strategies to treat and prevent botulism. Here, details on the molecular cloning, protein expression, purification, and immunoreactivity of BoNT/F domains from Clostridium botulinum are presented. Initially, full-length synthetic genes encoding recombinant BoNT/F domains (catalytic, translocation, and receptor binding) were designed and cloned into Escherichia coli for expression. Recombinant proteins were then purified through GST affinity chromatography preceding elution of GST-free recombinant domains by thrombin protease. Soluble recombinant proteins encoding catalytic light chain and translocation N-terminal heavy chain were subsequently used to perform in vivo immunization. Polyclonal mouse antibodies specific to these domains were raised, confirmed by Western blot analysis and elevated immunoreactivity was identified through indirect ELISA. In conclusion, availability of the recombinant protein provides an effective system to study the immunological aspects of BoNT/F and corresponding applications in pathogen detection and vaccine candidacy.

Keywords: Clostridium botulunium; Botulinum Neurotoxin Type F (BoNT/F) domains; cloning; recombinant protein expression; immunoreactivity

DOI: http://dx.doi.org/10.3126/njb.v2i1.5634

Nepal Journal of Biotechnology Jan.2012, Vol.2(1): 1-15

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Author Biography

Bhupal Ban, Medy-Tox Inc., Cheongwon-gun, Chungbuk-do

Correspondence Author recent address: Microbiology Graduate Program, Department of Biology, New Mexico State University, Las Cruces, NM 88003, United States of America

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How to Cite

Dulal, S., Ban, B., Yang, G. H., & Jung, H. H. (2012). Cloning, Expression, Purification, and Characterization of<i> Clostridium botulinum</i> Neurotoxin Serotype F Domains. Nepal Journal of Biotechnology, 2(1), 1–15. https://doi.org/10.3126/njb.v2i1.5634

Issue

Vol. 2 No. 1 (2012)

Section

Original Research Articles

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