title: Molecular cloning and functional analysis of a recombinant ribosome-inactivating protein (alpha-momorcharin) from Momordica charantia.
abstract: Alpha-momorcharin (α-MC), a member of the ribosome-inactivating protein (RIP) family, has been used not only as antiviral, antimicrobial, and antitumor agents, but also as toxicant to protozoa, insects, and fungi. In this study, we expressed the protein in Escherichia coli Rosetta (DE3) pLysS strain and purified it by nickel-nitrilotriacetic acid affinity chromatography. A total of 85 mg of homogeneous protein was obtained from 1 l culture supernatant of Rosetta (DE3) pLysS, showing a high recovery rate of 73.9%. Protein activity assay indicated that α-MC had both N-glycosidase activity and DNA-nuclease activity, the former releasing RIP diagnostic RNA fragment (Endo's fragment) from rice rRNAs and the latter converting supercoiled circular DNA of plasmid pET-32a(+) into linear conformations in a concentration-dependent manner. Specially, we found that α-MC could inhibit the mycelial growth of Fusarium solani and Fusarium oxysporum with IC(50) values of 6.23 and 4.15 μM, respectively. Results of optical microscopy and transmission electron microscopy demonstrated that α-MC caused extensive septum formation, loss of integrity of the cell wall, separation of the cytoplasm from the cell wall, deformation of cells with irregular budding sites, and apoptosis in F. solani. Moreover, α-MC was active against Pseudomonas aeruginosa with an IC(50) value of 0.59 μM. The α-MC protein carries a high potential for the design of new antifungal drugs or the development of transgenic crops resistant to pathogens.
authors.affiliation: State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan 430072, Hubei Province, People's Republic of China.
authors.forename: Shuzhen
authors.initials: S
authors.lastname: Wang
authors.forename: Yubo
authors.initials: Y
authors.lastname: Zhang
authors.forename: Honggao
authors.initials: H
authors.lastname: Liu
authors.forename: Ying
authors.initials: Y
authors.lastname: He
authors.forename: Junjie
authors.initials: J
authors.lastname: Yan
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Wu
authors.forename: Yi
authors.initials: Y
authors.lastname: Ding
mesh_terms: D003001:Cloning, Molecular; D003851:Deoxyribonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D005670:Fusarium / Q000187:drug effects / Q000254:growth & development; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D029777:Momordica charantia / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000254:growth & development; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D054788:Ribosome Inactivating Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; C039582:MMC protein, Momordica charantia; D003851:Deoxyribonucleases; D006026:Glycoside Hydrolases; D054788:Ribosome Inactivating Proteins