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 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 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 endow peptidase 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. (C) 2002 Elsevier Science (USA). All rights reserved.