The activity of the light (L) chain of tetanus toxin, and of mutants constructed by site-directed mutagenesis, was studied by expression and purification of the proteins from E. coli. Wild-type recombinant L chain (pTet87) was active in the inhibition of exocytosis from cultured bovine adrenal chromaffin cells, although at a level 5–15% of that of L chain purified from tetanus toxin. L chain mutants which terminated at Leu-438 (pTet89), or which contained a Cys-to-Ser mutation at residue 439 (pTet88) were equally as active as the full-length recombinant protein. The reduced activity of pTet87 L chain correlated with C-terminal proteolysis of the protein upon purification. A tryptic fragment derived from native light chain and which terminated at Leu-434 also showed reduced activity in the exocytosis assay, consistent with a requirement of the C-terminal region of the L chain for maximal activity. pTet87 L chain, but neither of the mutants, could be associated with purified H (heavy) chain to form a covalent dimer which induced the symptoms of tetanus in mice. The ability to form biologically active toxin using recombinant L chain will be of great value in structure-function studies of tetanus toxin.
