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 cloning of the L chain of TeNT from Clostridium tetani strain Y-IV-3 (WS 15) and its expression in Escherichia coli as a glutathione S-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.
Dipartimento di Scienze Biomediche, Università di Padova, Padova, I-35121, Italy.
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Grandi
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Peggion
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Montecucco
D003001:Cloning, Molecular; D003460:Crystallization; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013744:Tetanus Toxin / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D008565:Membrane Proteins; D010446:Peptide Fragments; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; D013744:Tetanus Toxin; D008666:Metalloendopeptidases; C093556:zinc-endopeptidase, tetanus neurotoxin