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

• Published in: Biotechnology and applied biochemistry 2001-10, Vol.34 (2), p.85-92
• Main Authors: PHAN VAN CHI, HOANG QUOC TRUONG, NGUYEN THUY HA, CHUNG, Won-Ii, LE TRAN BINH
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Base Sequence; Biological and medical sciences; Biotechnology; Cucurbitaceae; Escherichia coli - enzymology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Genes, Plant; Mission oriented research; Molecular Sequence Data; N-Glycosyl Hydrolases - metabolism; Plant Proteins - biosynthesis; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - pharmacology; Prospectives; Protein Biosynthesis - drug effects; Protein Synthesis Inhibitors - chemistry; Protein Synthesis Inhibitors - isolation & purification; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - pharmacology; Ribosome Inactivating Proteins; ribosome-inactivating protein; RNA, Ribosomal - antagonists & inhibitors; RNA, Ribosomal - biosynthesis; RNA, Ribosomal - metabolism; trichobakin; Trichosanthes; Trichosanthes - enzymology; trichosanthin
• ISSN: 0885-4513; 1470-8744
• DOI: 10.1042/ba20010026

We have isolated a genomic clone encoding trichobakin (TBK), a type I ribosome-inactivating protein from the plant Trichosanthes sp. Bac Kan 8-98 (family Cucurbitaceae), by PCR using specific primers designed from the cDNA sequences of alpha-trichosanthin. The sequence encoding mature TBK was constructed in the pET-21d(+) vector for overexpression in Escherichia coli strain BL21(DE3). The recombinant protein was purified to homogeneity by CM-Sepharose chromatography on FPLC with a final yield of about 55 mg/l of culture. The protein has a molecular mass of about 27 kDa, as shown by SDS/PAGE and matrix-assisted laser-desorption ionization MS. It was found that the protein inhibited luciferase mRNA translation in the rabbit reticulocyte cell-free system with an IC(50) value (that which causes a 50% reduction of residual translational activity) of about 3.5 pM. The rRNA N-glycosidase activity of the protein was also proved at the above-mentioned concentration after rRNAs were treated with acid aniline.