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.

[1]  S. Swaminathan,et al.  Structure of the receptor binding fragment HC of tetanus neurotoxin , 1997, Nature Structural Biology.

[2]  G. Schiavo,et al.  Metal substitution of tetanus neurotoxin. , 1997, The Biochemical journal.

[3]  L. Furenlid,et al.  X-ray absorption spectroscopy study of zinc coordination in tetanus neurotoxin, astacin, alkaline protease and thermolysin. , 1996, European journal of biochemistry.

[4]  G. Schiavo,et al.  Structure and function of tetanus and botulinum neurotoxins , 1995, Quarterly Reviews of Biophysics.

[5]  G. Schiavo,et al.  Structural studies on the zinc-endopeptidase light chain of tetanus neurotoxin. , 1995, European journal of biochemistry.

[6]  G. Schiavo,et al.  [39] Tetanus and botulism neurotoxins: Isolation and assay , 1995 .

[7]  F. Gasse,et al.  The present status of tetanus and tetanus vaccination. , 1995, Current topics in microbiology and immunology.

[8]  N. Fairweather,et al.  Production of biologically active light chain of tetanus toxin in Escherichia coli , 1993, FEBS letters.

[9]  F. Benfenati,et al.  Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin , 1992, Nature.

[10]  L. Tauc,et al.  Minimal essential domains specifying toxicity of the light chains of tetanus toxin and botulinum neurotoxin type A. , 1992, The Journal of biological chemistry.

[11]  A. Henschen,et al.  Limited proteolysis of tetanus toxin. Relation to activity and identification of cleavage sites. , 1991, European journal of biochemistry.

[12]  P. V. von Hippel,et al.  Calculation of protein extinction coefficients from amino acid sequence data. , 1989, Analytical biochemistry.

[13]  L. Simpson.  Botulinum neurotoxin and tetanus toxin , 1989 .

[14]  A. Henschen,et al.  Tetanus toxin: primary structure, expression in E. coli, and homology with botulinum toxins. , 1986, The EMBO journal.

[15]  N. Fairweather,et al.  The complete nucleotide sequence of tetanus toxin. , 1986, Nucleic acids research.

[16]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[17]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.