Cloning and expression of functional fragment C of tetanus toxin

Abstract

A segment of Clostridium tetani DNA corresponding to fragment C of tetanus toxin was amplified by using the polymerase chain reaction. This fragment was cloned into expression vector pTTQ8, under the control of the tac promoter. Expression of this plasmid in Escherichia coli resulted in the production of a protein consisting of 8 amino acids of the vector fused to the C-terminal 460 amino acids of tetanus toxin. This protein (rFragment C) was recognized by an antipeptide antibody specific for fragment C in an enzyme-linked immunosorbent assay and on immunoblots. rFragment C could be purified significantly in one step by immunoaffinity chromatography. Immunization of mice with rFragment C resulted in the production of antibodies that were able to protect the mice against a challenge with tetanus toxin. rFragment C bound to ganglioside GT1b and to neuronal cells in a manner indistinguishable from that of fragment C obtained by papain cleavage of tetanus toxin. For many applications, rFragment C appears to be a suitable alternative to tetanus toxin or toxin-derived fragment C.