No abstract

The amino acid sequence of the first 30 residues of fragment C of tetanus toxin was determined, and a mixture of 32 complementary oligonucleotides, each 17 bases long, was synthesized. A 2-kilobase (kb) EcoI fragment of Clostridium tetani DNA was identified by Southern blotting and was cloned into the Escherichia coli plasmid vector pAT153 with the 32P-labeled oligonucleotide mixture as a probe. A second 3.2-kb BglII fragment was identified and cloned with the 2-kb EcoRI fragment as a probe. The nucleotide sequence of 1.8 kb of this DNA was determined and was shown to encode the entire fragment C and a portion of fragment B of tetanus toxin. The tetanus DNA was expressed in E. coli with pWRL507, a plasmid vector containing the trp promoter and a portion of the trpE gene. The trpE-tetanus fusion proteins were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were shown to react with anti-fragment C antibody.Tetanus toxin is a potent inhibitor of the central nervous system. It causes spastic paralysis by blocking the release of inhibitory transmitters from inhibitory synapses (for a review, see reference 1). The exact mode of action of tetanus toxin at the molecular level remains unknown. The toxin, produced by Clostridium tetani, is synthesized as a 150,000-dalton polypeptide (Fig. 1). Upon lysis of the bacterium, the toxin is released from the cells, concomitantly with proteolytic cleavage of the molecule by an endogenous protease. The resulting molecule, termed "extracellular toxin," is composed of two fragments designated the light and heavy chains (13). These chains are held together by one or more disulfide bonds. Purified heavy and light chains are by themselves virtually nontoxic, but when they are reassociated, toxicity is restored.Papain digesion of tetanus toxin results in cleavage of the heavy chain to give two fragments, B and C (8) (Fig. 1). Purified fragment C is completely nontoxic in animals, whereas fragment B retains some residual toxicity at high doses, although this activity is manifest as a flaccid paralysis in mice rather than the spastic paralysis characteristic of tetanus toxin (7,8).Immunity to tetanus toxin is provided by the administration of formaldehyde-treated toxin (tetanus toxoid). Fragments B and C have also been used to successfully immunize animals against tetanus, indicating that the entire molecule is not essential for protection (6). However, the preparation of these fragments is time-consuming and not commercially feasible. Studies with monoclonal antibodies have also shown that all three domains of the tetanus toxin molecule (the light chain, the amino-terminal half of the heavy chain, and the C fragment) may induce neutralizing antibodies (10, 25).To characterize tetanus toxin further at the molecular level, we undertook the cloning, nucleotide sequencing, and expression in Escherichia coli of DNA encoding fragment C of tetanus toxin. We used synthetic oligonucleotides com-* Corresponding author.plementary to the amino acid sequence of fragment C ...

Two recombinant plasmids, pTetl1 and pTet18, which express nontoxic protein fragments of tetanus toxin in Escherichia coli, were constructed. pTetlI protein (86 kilodaltons) is a fusion between part of the E. coli trpE protein and 441 amino acids of tetanus fragment C. and pTetl8 (63 kilodaltons) consists of part of fragment B and all of fragment C of tetanus toxin. The synthesis of these proteins was induced in E. coli cultures, and the proteins were partially purified. Mice were immunized with these proteins, and dose-dependent titers of anti-tetanus toxoid antibodies were obtained. The proteins were able to induce neutralizing antibodies in mice, as demonstrated by the ability of mice immunized with 1 ,ig or more of protein to survive challenge with 10 50% lethal doses of tetanus toxin.