High-level expression of tetanus toxin fragment C-thioredoxin fusion protein in Escherichia coli

An insert of Clostridium tetani DNA corresponding to fragment C of tetanus toxin was amplified by PCR. This 1.4 kb fragment was cloned into the high-e xpression vector pET32a, under control of the T7 promoter. Expression of th is plasmid in Escherichia coli BL21 (DE3) resulted in the production of a f usion protein ( approximate to 62 kDa) consisting of 112 amino acids of thi oredoxin and approximate to 450 amino acids of fragment C. This fusion prot ein was recognized by anti-tetanus toroid antiserum in an ELISA and on immu noblots. The recombinant fragment-C-thioredoxin protein was purified signif icantly in one step by Ni2+-chelate Sepharose, the final yield being approx imate to 35 mg/l. Immunization of animals with the recombinant protein prod uced antibodies that were able to recognize the tetanus toxin. By using thi s gene-fusion expression system we produced soluble fragment C of tetanus t oxin in a high yield, preventing many problems inherent in the use of other expression systems that produce either insoluble fragment C in inclusion b odies, or a soluble form, but in low yield, using E. coli as the expression host.