Preparation of specifically activatable endopeptidase derivatives of Clostridium botulinum toxins type A, B, and C and their applications.
Clostridium botulinum neurotoxins are potently toxic proteins of 150 kDa with specific endopeptidase activity for SNARE proteins involved in vesicle docking and release. Following treatment with trypsin, a fragment of botulinum neurotoxin serotype A that lacks the C-terminal domain responsible for neuronal cell binding, but retains full catalytic activity, can be obtained. Known as the LH(N) fragment, we report the development of a recombinant expression and purification scheme for the isolation of comparable fragments of neurotoxin serotypes B and C. Expressed as maltose-binding protein fusions, both have specific proteolytic sites present between the fusion tag and the light chain to facilitate removal of the fusion, and between the light chain endopeptidase and the H(N) translocation domains to facilitate activation of the single polypeptide. We have also used this approach to prepare a new variant of LH(N)/A with a specific activation site that avoids the need to use trypsin. All three LH(N)s are enzymatically active and are of low toxicity. The production of specifically activatable LH(N)/A, LH(N)/B, and LH(N)/C extends the opportunities for exploitation of neurotoxin fragments. The potential utility of these fragments is discussed.
Sutton
J Mark
JM
Health Protection Agency, Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 0JG, UK.
Wayne
Jonathan
J
Scott-Tucker
Anthony
A
O'Brien
Susan M
SM
Marks
Philip M H
PM
Alexander
Frances C G
FC
Shone
Clifford C
CC
Chaddock
John A
JA
D000818:Animals; D001483:Base Sequence; D001905:Botulinum Toxins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003014:Clostridium botulinum / Q000201:enzymology / Q000235:genetics; D010450:Endopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D015870:Gene Expression; D005822:Genetic Vectors; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
D010446:Peptide Fragments; D011994:Recombinant Proteins; C096323:rimabotulinumtoxinB; D010450:Endopeptidases; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; C096324:botulinum toxin type C