Cloning and expression of tetanus toxin fragment C in E-coli

The fragment C of tetanus toxin was amplified from Clostridium tetani DNA by PCR. This fragment was cloned into expression vector pET-28a(+),under the control of the T7 promoter. Expression of this plasmid in E.coli resulted in the production of a protein consisting of 6 x His of the vector fused to the N-terminal 451 amino acids of tetanus toxin. After induction with 1 mmol/L IPTG, TTC was expressed in E. coli BL21(DE3). The protein product accounted for 8.2% of the bacteria total protein in soluble form, SDS-PAGE and Western blot analysis of TTC recombinant protein confirmed this result. The expression products were also purified by Ni2+-IDA-Sephrose 6B column. Immunization of mice with rTTC resulted in the production of antibodies that were able to protect mice against a challenge with tetanus toxin; furthermore, rTTC in vivo appeared to be able to undergo retrograde axonal transport.