Expression and purification of catalytically active, non-toxic endopeptidase derivatives of clostridium botulinum toxin type A

John A. Chaddock, Michael H. Herbert, Roger J. Ling, Frances C.G. Alexander, Sarah J. Fooks, Dean F. Revell, Conrad P. Quinn, Clifford Shone, Keith A. Foster

Research output: Contribution to journalArticlepeer-review

61 Citations (Scopus)

Abstract

Clostridium botulinum neurotoxin type A is a potently toxic protein of 150 kDa with specific endopeptidase activity for the SNARE protein SNAP-25. Proteolytic cleavage of BoNT/A with trypsin leads to removal of the C-terminal domain responsible for neuronal cell binding. Removal of this domain result in a catalytically active, non-cell-binding derivative termed LHN/A. We have developed a purification scheme to prepare LHN/A essentially free of contaminating BoNT/A. LHN/A prepared by this scheme retains full enzymatic activity, is stable in solution, and is of low toxicity as demonstrated in a mouse toxicity assay. In addition, LHN/A has minimal e+ect on release of neurotransmitter from a primary cell culture model. Both the mouse bioassay and in vitro release assay suggest BoNT/A is present at less than 1 in 106 molecules of LHN/A. This represents a significant improvement on previously reported figures for LHN/A, and also the light chain domain, previously purified from BoNT/A. To complement the preparation of LHN/A from holotoxin, DNA encoding LHN/A has been introduced into Escherichia coli to facilitate expression of recombinant product. Expression and purification parameters have been developed to enable isolation of soluble, stable endopeptidase with a toxicity profile enhanced on that of LHN/A purified from BoNT/A. The recombinant-derived material has been used to prepare antisera that neutralise a BoNT/A challenge. The production of essentially BoNT/A-free LHN/A by two different methods and the possibilities for exploitation are discussed.

Original languageEnglish
Pages (from-to)219-228
Number of pages10
JournalProtein Expression and Purification
Volume25
Issue number2
DOIs
Publication statusPublished - 2002

Bibliographical note

Funding Information:
We are grateful to I. McIntee for supply of cell paste and M.J. Duggan, J.R. Purkiss, and N. Allison for helpful discussion. We also wish to thank B. Hallis for kindly supplying antibodies to BoNT/A and to R. Fretwell and S. Doward for providing data. This work was initially supported by Speywood Pharmaceuticals and latterly by Allergan Inc.

Copyright:
Copyright 2018 Elsevier B.V., All rights reserved.

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