Expression and purification of catalytically active, non-toxic endopeptidase derivatives of Clostridium botulinum toxin type A.
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.
Chaddock
John A
JA
Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 0JG, UK. john.chaddock@camr.org.uk
Herbert
Michael H
MH
Ling
Roger J
RJ
Alexander
Frances C G
FC
Fooks
Sarah J
SJ
Revell
Dean F
DF
Quinn
Conrad P
CP
Shone
Clifford C
CC
Foster
Keith A
KA
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D015153:Blotting, Western; D019274:Botulinum Toxins, Type A / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D002384:Catalysis; D003014:Clostridium botulinum / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D051379:Mice; D009465:Neuromuscular Agents / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D018377:Neurotransmitter Agents / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology
D009465:Neuromuscular Agents; D018377:Neurotransmitter Agents; D011994:Recombinant Proteins; D010450:Endopeptidases; D019274:Botulinum Toxins, Type A