title: Bacterial expression and enzymatic activity analysis of ME1, a ribosome-inactivating protein from Mirabilis expansa.
abstract: Ribosome-inactivating proteins (RIPs) are toxic proteins synthesized by many plants and some bacteria, that specifically depurinate the 28S RNA and thus interrupt protein translation. RIPs hold broad interest because of their potential use as plant defense factors against pathogens. However, study of the activity of type I RIPs has been hampered since their expression in Escherichia coli has typically been toxic to the model system. Mirabilis expansa, an Andean root crop, produces a type I RIP called ME1 in large quantities in its storage roots. In this study, the cDNA sequence of ME1 was used to successfully express the recombinant ME1 protein in E. coli. The production of recombinant ME1 in E. coli was confirmed by Western blot analysis using anti-ME1 antibodies. The studies with fluorescence-labeled ME1 showed that ME1 can enter bacteria and be distributed in the cytoplasm uniformly, indicating its ability to access the protein synthesis machinery of the bacteria. The recombinant enzyme was active and depurinated yeast ribosomes. However, both native and recombinant ME1 proteins failed to depurinate the E. coli ribosomes, explaining the non-toxicity of recombinant ME1 to E. coli. Structural modeling of ME1 showed that it has folding patterns similar to other RIPs, indicating that ME1 and PAP, which share a similar folding pattern, can show different substrate specificity towards E. coli ribosomes. The results presented here are very significant, as few reports are available in the area of bacterial interaction with type I RIPs.
authors.affiliation: Department of Medicine, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206, USA.
authors.forename: Ramarao
authors.initials: R
authors.lastname: Vepachedu
authors.forename: Sang-Wook
authors.initials: SW
authors.lastname: Park
authors.forename: Neelam
authors.initials: N
authors.lastname: Sharma
authors.forename: Jorge M
authors.initials: JM
authors.lastname: Vivanco
mesh_terms: D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D039282:Mirabilis / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D018517:Plant Roots / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D054788:Ribosome Inactivating Proteins
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D009699:N-Glycosyl Hydrolases; D054788:Ribosome Inactivating Proteins