Molecular cloning and functional analysis of a recombinant ribosome-inactivating protein (alpha-momorcharin) from Momordica charantia

Alpha-momorcharin (alpha-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 alpha-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 alpha-MC could inhibit the mycelial growth of Fusarium solani and Fusarium oxysporum with IC50 values of 6.23 and 4.15 mu M, respectively. Results of optical microscopy and transmission electron microscopy demonstrated that alpha-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, alpha-MC was active against Pseudomonas aeruginosa with an IC50 value of 0.59 mu M. The alpha-MC protein carries a high potential for the design of new antifungal drugs or the development of transgenic crops resistant to pathogens.