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 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.
Chi
P V
PV
Institute of Biotechnology, National Center for Natural Science and Technology, Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam. pvchi@netnam.vn
Truong
H Q
HQ
Ha
N T
NT
Chung
W I
WI
Binh
L T
LT
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D017343:Genes, Plant; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D012335:RNA, Ribosomal / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology; D054788:Ribosome Inactivating Proteins; D029778:Trichosanthes / Q000201:enzymology*
D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D012335:RNA, Ribosomal; D011994:Recombinant Proteins; D009699:N-Glycosyl Hydrolases; D054788:Ribosome Inactivating Proteins; C444621:TBK protein, Trichosanthes