PRODUCTION OF BIOLOGICALLY-ACTIVE LIGHT-CHAIN OF TETANUS TOXIN IN ESCHERICHIA-COLI - EVIDENCE FOR THE IMPORTANCE OF THE C-TERMINAL 16 AMINO-ACIDS FOR FULL BIOLOGICAL-ACTIVITY

The activity of the light (L) chain of tetanus toxin, and of mutants c onstructed by site-directed mutagenesis, was studied by expression and purification of the proteins from E. coli. Wild-type recombinant L ch ain (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 resid ue 439 (pTet88) were equally as active as the full-length recombinant protein. The reduced activity of pTet87 L chain correlated with C-term inal proteolysis of the protein upon purification. A tryptic fragment derived from native light chain and which terminated at Leu434 also sh owed reduced activity in the exocytosis assay, consistent with a requi rement of the C-terminal region of the L chain for maximal activity. p Tet87 L chain, but neither of the mutants, could be associated with pu rified H (heavy) chain to form a covalent dimer which induced the symp toms of tetanus in mice. The ability to form biologically active toxin using recombinant L chain will be of great value in structure-functio n studies of tetanus toxin.