Analysis of the substrate recognition domain determinants of Botulinum Type B toxin using Phage Display

E. R. Evans*, J. M. Sutton, A. Gravett, Clifford Shone

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    10 Citations (Scopus)


    The botulinum neurotoxin endopeptidases appear to recognise their intracellular protein substrates via two distinct sites: the cleavage site sequence and a 'recognition site' motif. In the present study phage display has been employed to generate a library of vesicle-associated membrane protein (VAMP2) variants in which the toxin recognition motif (part of the SNARE motif ELDDRADA) has been modified. VAMP (1-94) was displayed on the surface of M13 bacteriophage and this fragment was recognised and cleaved by botulinum neurotoxin type B (BoNT/B). A phage-displayed library was constructed in which six residues of the recognition domain (VAMP residues 63-68; wild-type sequence LDDRAD) were randomised, and a selection method established for identifying cleaved VAMP variants. Sequence analysis of 24 clones revealed that 5 contained two acidic residues although none corresponded to the native sequence. Cleavage was reduced compared to wild-type VAMP, and cleavage of mutants containing no acidic residues was also observed. The data are discussed in relation to the substrate recognition mechanism of BoNT/B.

    Original languageEnglish
    Pages (from-to)446-453
    Number of pages8
    Issue number4
    Publication statusPublished - 15 Sept 2005


    • Clostridium botulinum neurotoxin type B
    • Phage display
    • SNARE motif
    • VAMP


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