[Construction, expression and purification of trichosanthin mutant gene TCS(FYY163-165CSA);]

To construct and express a trichosanthin (TCS) gene mutant and purify the expressed product in E.coli.

The potential antigenic determinant was predicted on TCS molecule by computer modeling and induced for site-directed mutation. The gene mutant TCS(FYY163-165CSA); was amplified by PCR using the genomic DNA of Trichosanthes kirilowii as a template and cloned into expression vector pRSET-A, then transfected into E.coli BL21 (DE3) for expression by inducing with IPTG. The expressed product was identified by Western blotting and purified by Ni-NTA affinity column chromatography.

The soluble target protein was successfully expressed in E.coli. Homogenous TCS mutant protein was obtained after purification of expressed product.

The site-directed mutagenesis, expression and purification of TCS provide a new approach for reconstructing TCS.