Characterization of trichobakin, a type I ribosome-inactivating protein from Trichosanthes sp Bac Kan 8-98

We have isolated a genomic clone encoding trichobakin (TBK), a type I ribos ome-inactivating protein from the plant Trichosanthes sp. Bac Kan 8-98 (fam ily Cucurbitaceae), by PCR using specific primers designed from the cDNA se quences of alpha -trichosanthin. The sequence encoding mature TBK was const ructed in the pET-21d(+) vector for overexpression in Escherichia coli stra in BL21(DE3). The recombinant protein was purified to homogeneity by CM-Sep harose chromatography on FPLC with a final yield of about 55 mg/l of cultur e. The protein has a molecular mass of about 27 kDa, as shown by SDS/PAGE a nd matrix-assisted laser-desorption ionization MS. It was found that the pr otein inhibited luciferase mRNA translation in the rabbit reticulocyte cell -free system with an IC50 value (that which causes a 50% reduction of resid ual translational activity) of about 3.5 pM. The rRNA glycosidase activity of the protein was also proved at the above-mentioned concentration after r RNAs were treated with acid aniline.