Analysis of the substrate recognition domain determinants of botulinum type B toxin using phage display
| Autor: | Clifford C. Shone, A. Gravett, E. R. Evans, John Mark Sutton |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Phage display
Botulinum Toxins Sequence analysis Vesicle-Associated Membrane Protein 2 Mutant Genetic Vectors Molecular Sequence Data Toxicology Cleavage (embryo) medicine.disease_cause Substrate Specificity Peptide Library Sequence Analysis Protein medicine Amino Acid Sequence Botulinum Toxins Type A Cloning Molecular DNA Primers M13 bacteriophage biology VAMP2 biology.organism_classification Molecular biology Protein Structure Tertiary Biochemistry Membrane protein Clostridium botulinum Bacteriophage M13 |
| Zdroj: | Toxicon : official journal of the International Society on Toxinology. 46(4) |
| ISSN: | 0041-0101 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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