P. Van Chi et al., Characterization of trichobakin, a type I ribosome-inactivating protein from Trichosanthes sp Bac Kan 8-98, BIOT APP B, 34, 2001, pp. 85-92
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.