Expression and Purification of Recombinant Trichosanthin in Escherichia Coli and Evaluation of Its Effect on HeLa Cell Proliferation

To clone TCS cDNA, construct pET-28a(+)-TCS plasmid, express and purify recombinant trichosanthin(rTCS) from E.coli, and evaluat the effect on proliferation of HeLa cells. Methods TCS cDNA was amplified by RT-PCR and then cloned into pET-28a(+) vector. pET-28a(+)-TCS was then transformed into E.coli BL21(DE3) and rTCS was expressed by IPTG induction and purified by Ni-NTA resin. MTT assay was performed to detect the effects of rTCS and nTCS on proliferation of HeLa cells after 24h, 48h or 72h treatments. Results 1. TCS cDNA was successfully cloned and inserted into the pET-28a(+)vector; 2. The rTCS was expressed by IPTG induction in BL21(DE3) and most of rTCS was found to mainly exist in the inclusion body. Expression level of rTCS was positively correlated with the inducing time within 0~8h; 3. The rTCS was purified from BL21(DE3) induced by IPTG for 8h by Ni-NTA resin; 4. MTT assay demonstrated that both rTCS and nTCS can inhibit proliferation of HeLa cells in concentration- and time-dependent manner(P<0.05),and the IC₅₀ value of rTCS was less than that of nTCS. Conclusion The TCS cDNA was cloned, plasmid pET-28a(+)-TCS was constructed and rTCS protein was successfully expressed and purified from BL21(DE3). The rTCS was less cytotoxic than nTCS against HeLa cancer cells in vitro(P<0.05).