A recombinant ribosome‐inactivating protein from the plant Phytolacca dioica L. produced from a synthetic gene

FEBS Letters

Scognamiglio, Roberta¹  Di Maro, Antimo¹  Siniscalco, Gesualdo³  Del Vecchio Blanco, Francesca²  Parente, Augusto²  Di Donato, Alberto¹  Cafaro, Valeria¹ 

[1] Dipartimento di Chimica Organica e Biologica, Università Federico II di Napoli, Via Mezzocannone, 16, 80134 Naples, Italy;Dipartimento di Scienza della Vita, Seconda Università di Napoli, Caserta, Italy;Dipartimento di Biologia Vegetale, Università Federico II di Napoli, Via Foria, Naples, Italy

关键词: Ribosome-inactivating protein;    Protein engineering;    Toxin;    RIP;    ribosome-inactivating protein;    PD-L;    Phytolacca dioica leaf RIP;    PD-S;    Phytolacca dioica seed RIP;    EDTA;    ethylenediaminetetraacetic acid;    PAGE;    polyacrylamide gel electrophoresis;    SDS;    sodium dodecylsulfate;    Tris;    tris(hydroxymethyl)aminomethane;    DTT;    dithiothreitol;    HPLC;    high pressure liquid chromatography;    IPTG;    isopropyl-β-d-thiogalactopyranoside;    dNTP;    deoxynucleotide triphosphate;    ATP;    adenosine triphosphate;   

DOI  :  10.1016/S0014-5793(98)01240-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Phytolacca dioica L. leaves produce at least two type-I ribosome-inactivating proteins. Each polypeptide chain is subjected to different post-translational modifications giving rise to PD-L1 and PD-L2, and PD-L3 and PD-L4, each polypeptide pair having the same primary structure. With the aim of exploiting the cytotoxic properties of these proteins as potential biological phytodrugs, a gene encoding PD-L4 was designed based on criteria expected to maximize the translation efficiency in tomato. The gene was constructed from 18 oligonucleotides and preliminarily expressed in Escherichia coli, using the T7 promoter system. The protein produced was insoluble and accumulated in inclusion bodies to about 300 mg/l of culture. Ribosome-inactivating activity was generated by controlled oxidation of the reduced and denatured protein. The recombinant protein was indistinguishable from natural PD-L4 as isolated from leaves of Phytolacca dioica, in both catalytic activity and primary structure.

【 授权许可】

Unknown