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Biotechnology and Applied Biochemistry (2001) 34, (85–92) (Printed in Great Britain)

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

Phan Van Chi*¹, Hoang Quoc Truong*, Nguyen Thuy Ha*, Won-Il Chung† and Le Tran Binh*

*Institute of Biotechnology (IBT ), National Center for Natural Science and Technology (NCST ), Hoang Quoc Viet Road, Cau Giay, Hanoi, Vietnam, and †Korea Advanced Institute of Science & Technology (KAIST ), 373-1 Kusong-dong, Yusong-ku, Taejon, 305-701, Korea

Key words: expression, purification, RIP, rRNA N-glycosidase, TBK.

Abbreviations used: RIP, ribosome-inactivating protein; TBK, trichobakin; IPTG, isopropyl b-d-thiogalactoside; MALDI-MS, matrix-assisted laser-desorption ionization MS.

¹To whom correspondence should be addressed (e-mail pvchi@netnam.vn).

The nucleotide sequence data reported have been submitted to the DDBJ, GenBank and EMBL Nucleotide Sequence Databases under the accession number AB039324.

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 a-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 IC₅₀ 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.

Received 8 May 2001; accepted 7 July 2001

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