title: Abrin-a A chain expressed as soluble form in Escherichia coli from a PCR-synthesized gene is catalytically and functionally active.
abstract: Abrin-a A chain (ABRaA) is a potent plant toxin, which possesses N-glycosylase activity toward eukaryotic 28S rRNA, and may have potential use in cancer therapy. To improve levels of expression in Escherichia coli, the gene encoding ABRaA was optimized by replacing rare codons with high-frequency ones, and synthesized using two-step PCR. The optimized ABRaA was cloned into the pET-His vector, and highly expressed in cytoplasm of E. coli. The yield of the purified recombinant (r) ABRaA proteins was up to 80 mg/l of induced culture. The rABRaA was one-step purified to homogeneity and its RNA-N-glycosylase ability to inhibit protein biosynthesis in a cell-free system and to depurinate 28S rRNA in rat liver ribosomes was demonstrated in vitro. The MTT assay showed that it also had a killing effect on human hepatoma cell line SMMC-7721 and myeloma cell line Sp2/0. For the first time, ABRaA expressed as soluble form in E. coli from a PCR-synthesized gene is catalytically and functionally active.
authors.affiliation: The Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, No. 20 Dongda Street, Fengtai District, Beijing 100071, China.
authors.forename: Li-Chun
authors.initials: LC
authors.lastname: Wang
authors.forename: Lin
authors.initials: L
authors.lastname: Kang
authors.forename: Ting-Mao
authors.initials: TM
authors.lastname: Hu
authors.forename: Jing-Lin
authors.initials: JL
authors.lastname: Wang
mesh_terms: D000036:Abrin / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology; D002384:Catalysis; D002474:Cell-Free System; D003593:Cytoplasm / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008099:Liver; D009101:Multiple Myeloma / Q000378:metabolism / Q000473:pathology; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012339:RNA, Ribosomal, 28S / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D054788:Ribosome Inactivating Proteins; D012270:Ribosomes / Q000378:metabolism
chemical_list: D012339:RNA, Ribosomal, 28S; D011994:Recombinant Proteins; D000036:Abrin; D009699:N-Glycosyl Hydrolases; D054788:Ribosome Inactivating Proteins