Expression and characterisation of the heavy chain of tetanus toxin: Reconstitution of the fully-recombinant dichain protein in active form

Tetanus toxin, composed of a disulphide-linked heavy (HC) and light (LC) ch ain, preferentially blocks the release of inhibitory neurotransmitters in t he spinal cord by Zn2+-dependent proteolytic cleavage of synaptobrevin. Thi s intoxication involves binding via HC to ecto-acceptors on peripheral nerv e endings, followed by internalisation and retrograde transportation to its prime site of action in central neurons. To facilitate exploitation of the toxin's unique activities, HC was expressed at a high level in Escherichia coil as a fusion with maltose binding protein; after cleavage by thrombin, free HC was isolated and its identity confirmed by Western blotting and N- terminal microsequencing, The expressed and native HC gave very similar cir cular dichroism spectra, excluding any gross differences in their folded st ructures. Recombinant HC antagonised the neuromuscular paralysing activity of the native toxin, by competing for binding to neuronal ecto-acceptors. T he HC was reconstituted with bacterially-expressed LC to create disulphide- bridged dichain toxin that blocked neuromuscular transmission. The fully-re combinant toxin produced spastic paralysis in mice characteristic of the bl ockade of central inhibitory synapses, revealing that it undergoes axonal t ransport to the spinal cord, like the native toxin but with a reduced effic acy. This first report of the large-scale production of recombinant tetanus toxin in active form should facilitate studies on the use of engineered in nocuous forms of the toxin as neuronal transport vehicles.