RESUMO
AGG, the codon of 122 Arg of trichosanthin (TCS) was mutated to GGG (Gly) by U-DNA site-directed mutagenesis. The mutant TCS was expressed and purified from the supernatant of host cells, its activities were assayed and compared with expressed wild-type TCS. The results showed that the R122G TCS was still active as a RNA N-glycosidase but its ability to inhibit protein synthesis was 160-fold less than that of wild-type TCS, its abortifacient ability on mid-term gestated mice reduced from 100% to 9.3%, which suggested that 122 Arg was indirectly involved in the catalysis, and it played an important role in its bioactivities.
RESUMO
Western blot result showed that T(8)C(12), an anti-trichosanthin (TCS) monoclonal antibody, could bind to a CNBr-cleaved TCS fragment with MW of 8 kD. The epitope was located in 1-72 of the N terminal of TCS as shown by amino acid analysis. A random 6-aa peptide library cloned in pIII of phage M13 was screened by T(8)C(12). After two cycles screening, 15 positive clones were randomly selected and six kinds of 6-aa sequences were determined, which showed to be highly homologous with a Ser/Thr-(X)-(X)-Arg motif, the X denoting hydrophobic amino acids. The motif was found to be similar to the first 3-5 amino acid sequence of the TCS N-terminal. The synthesized 1-8 peptide of TCS showed competitive binding to T(8)C(12) with native TCS. It suggested that 3-5 amino acid residues of TCS N-terminal was the core of epitope recognized by T(8)C(12).