Reconstitution of Diphtheria Toxin from Two Nontoxic Cross-Reacting Mutant Proteins

The isolation of a new type of mutant Corynephage β, which carries a missense mutation in the structural gene for diphtheria toxin synthesis is described. The lysogenic C78(β197)tox-crm+ strain of Corynebacterium diphtheriae produces a nontoxic, extracellular protein of molecular weight 62,000. This protein is immunologically indistinguishable from toxin itself but inhibits the action of toxin on HeLa cells, probably by competing for attachment sites on the cell membrane. In contrast to fragment A derived from diphtheria toxin, fragment A197 is unable to catalyze the inactivation of eucaryotic polypeptidyl-transfer RNA-transferase II. When mixtures of the two nontoxic mutant proteins, enzymically active crm45 protein and enzymically inactive crm197 protein, are subjected to mild treatment with trypsin in the presence of a thiol and then allowed to reoxidize after dialysis to remove excess thiol, "diphtheria toxin" is reconstituted in high yield.