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) chain, preferentially blocks the release of inhibitory neurotransmitters in the spinal cord by Zn2+-dependent proteolytic cleavage of synaptobrevin. This intoxication involves binding via HC to ecto-acceptors on peripheral nerve 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 coli 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 circular dichroism spectra, excluding any gross differences in their folded structures. Recombinant HC antagonised the neuromuscular paralysing activity of the native toxin, by competing for binding to neuronal ecto-acceptors. The HC was reconstituted with bacterially-expressed LC to create disulphide-bridged dichain toxin that blocked neuromuscular transmission. The fully-recombinant toxin produced spastic paralysis in mice characteristic of the blockade of central inhibitory synapses, revealing that it undergoes axonal transport to the spinal cord, like the native toxin but with a reduced efficacy. This first report of the large-scale production of recombinant tetanus toxin in active form should facilitate studies on the use of engineered innocuous forms of the toxin as neuronal transport vehicles.
Li
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Department of Biochemistry, Imperial College, London, SW7 2AZ, UK.
Aoki
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Dolly
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D009469:Neuromuscular Junction / Q000187:drug effects; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D009435:Synaptic Transmission / Q000187:drug effects; D013744:Tetanus Toxin / Q000737:chemistry* / Q000235:genetics* / Q000494:pharmacology
D017931:DNA Primers; D004274:DNA, Recombinant; D011994:Recombinant Proteins; D013744:Tetanus Toxin