Recombinant and truncated tetanus neurotoxin light chain: Cloning, expression, purification, and proteolytic activity

Tetanus neurotoxin (TeNT) consists of two disulfide-linked polypeptide chai ns, heavy (H) and Light (L), The L chain is a zinc endopeptidase protein hi ghly specific for vesicle-associated membrane protein (VAMP), which is an e ssential component of the exocytosis apparatus. Here we describe the clonin g 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 pr otein. The full-length recombinant L chain, corresponding to residues 1-457 , was obtained as a mixture of proteins of slightly different mass with ide ntical N-terminal ends. To obtain a product useful for structural analysis and crystallization, a COOH-terminally truncated L chain (residues 1-427) w as cloned, expressed, and purified with high yield. This truncated L chain is more active than the full-length and wild-type proteins in the hydrolysi s of VAMP. Preliminary experiments of crystallization of the truncated reco mbinant L chain gave encouraging results. (C) 1999 Academic Press.