Expression and purification of catalytically active, non-toxic endopeptidase derivatives of Clostridium botulinum toxin type A.
Protein Expr Purif
; 25(2): 219-28, 2002 Jul.
Article
em En
| MEDLINE
| ID: mdl-12135553
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 LH(N)/A. We have developed a purification scheme to prepare LH(N)/A essentially free of contaminating BoNT/A. LH(N)/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, LH(N)/A has minimal effect 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 10(6) molecules of LH(N)/A. This represents a significant improvement on previously reported figures for LH(N)/A, and also the light chain domain, previously purified from BoNT/A. To complement the preparation of LH(N)/A from holotoxin, DNA encoding LH(N)/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 LH(N)/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 LH(N)/A by two different methods and the possibilities for exploitation are discussed.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Endopeptidases
/
Clostridium botulinum
/
Toxinas Botulínicas Tipo A
Limite:
Animals
Idioma:
En
Ano de publicação:
2002
Tipo de documento:
Article