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The Protein Journal

, Volume 26, Issue 1, pp 29–37 | Cite as

Cloning, Expression, Purification, and Characterization of Biologically Active Recombinant Hemagglutinin-33, Type A Botulinum Neurotoxin Associated Protein

  • Yu Zhou
  • Sowmya Paturi
  • Paul Lindo
  • Suzanne M. Shoesmith
  • Bal Ram Singh
Article

Abstract

Botulinum neurotoxin type A, the most toxic substance known to mankind, is produced by Clostridium botulinum type A as a complex with a group of neurotoxin-associated proteins (NAPs) through polycistronic expression of a clustered group of genes. Hemagglutinin-33 (Hn-33) is a 33 kDa subcomponent of NAPs, which is resistant to protease digestion, a feature likely to be involved in the protection of the botulinum neurotoxin from proteolysis. In order to fully understand the function of Hn-33, large amounts of Hn-33 will be needed without dealing with biosafety risks to grow large cultures of C. botulinum. There are difficulties to clone the genes with the high A + T contents produced by C. botulinum. We report here for the first time using the Gateway technology to clone functional Hn-33 that has been expressed in E. coli. The yield of the recombinant Hn-33 was about 12 mg per liter of E. coli culture. The recombinant Hn-33 folds well in aqueous solution as shown with circular dichroism spectra, resists temperature-denaturation, is totally resistant to trypsin proteolysis despite the presence of cleavage sites on the molecular surface, and maintains its biological activities comparable to the native Hn-33 hemagglutination.

Keywords

Botulinum neurotoxin circular dichroism spectroscopy hemagglutination assay hemagglutinin-33 trypsin digestion 

Notes

Acknowledgments

This work was supported by a grant from the US Army Medical Research and Material Command under Contract No. DAMD17-02-C-001 and by the National Institutes of Health through New England Center of Excellence for Biodefense (AI057159-01).

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Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Yu Zhou
    • 1
  • Sowmya Paturi
    • 1
  • Paul Lindo
    • 1
  • Suzanne M. Shoesmith
    • 1
  • Bal Ram Singh
    • 1
  1. 1.Department of Chemistry and Biochemistry, and Botulinum Research CenterUniversity of Massachusetts DartmouthNorth DartmouthUSA

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