Recombinant and Truncated Tetanus Neurotoxin Light Chain: Cloning, Expression, Purification, and Proteolytic Activity

Tetanus neurotoxin (TeNT) consists of two disulfide-linked polypeptide chains, heavy (H) and light (L). The L chain is a zinc endopeptidase protein highly specific for vesicle-associated membrane protein (VAMP), which is an essential component of the exocytosis apparatus. Here we describe the clonin...

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Bibliographic Details
Published in:Protein expression and purification 1999-03, Vol.15 (2), p.221-227
Main Authors: Tonello, Fiorella, Pellizzari, Rossella, Pasqualato, Sebastiano, Grandi, Guido, Peggion, Evaristo, Montecucco, Cesare
Format: Article
Language:English
Subjects:
Cloning, Molecular
Crystallization
crystals
Escherichia coli
Gene Expression
Humans
Membrane Proteins - metabolism
Metalloendopeptidases - biosynthesis
Metalloendopeptidases - genetics
Metalloendopeptidases - isolation & purification
Metalloendopeptidases - metabolism
metalloprotease
Peptide Fragments - biosynthesis
Peptide Fragments - genetics
Peptide Fragments - isolation & purification
Peptide Fragments - metabolism
R-SNARE Proteins
Recombinant Fusion Proteins - biosynthesis
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - isolation & purification
Recombinant Fusion Proteins - metabolism
Spectrometry, Fluorescence
Substrate Specificity
tetanus
tetanus neurotoxin
Tetanus Toxin - biosynthesis
Tetanus Toxin - genetics
Tetanus Toxin - isolation & purification
Tetanus Toxin - metabolism
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Summary:Tetanus neurotoxin (TeNT) consists of two disulfide-linked polypeptide chains, heavy (H) and light (L). The L chain is a zinc endopeptidase protein highly specific for vesicle-associated membrane protein (VAMP), which is an essential component of the exocytosis apparatus. Here we describe the cloning of the L chain of TeNT fromClostridium tetanistrain Y-IV-3 (WS 15) and its expression inEscherichia colias a glutathioneS-transferase fusion protein. The full-length recombinant L chain, corresponding to residues 1–457, was obtained as a mixture of proteins of slightly different mass with identical N-terminal ends. To obtain a product useful for structural analysis and crystallization, a COOH-terminally truncated L chain (residues 1–427) was cloned, expressed, and purified with high yield. This truncated L chain is more active than the full-length and wild-type proteins in the hydrolysis of VAMP. Preliminary experiments of crystallization of the truncated recombinant L chain gave encouraging results.
ISSN:1046-5928
1096-0279
DOI:10.1006/prep.1998.1007