title: Characterization of a highly potent antimicrobial peptide microcin N from uropathogenic Escherichia coli.
abstract: Microcin N is a low-molecular weight, highly active antimicrobial peptide produced by uropathogenic Escherichia coli In this study, the native peptide was expressed and purified from pGOB18 plasmid carrying E. coli in low yield. The pure peptide was characterized using mass spectrometry, N-terminal sequencing by Edman degradation as well as trypsin digestion. We found that the peptide is 74-residue long, cationic (+2 total charge), highly hydrophobic and consists of glycine as the first N-terminal residue. The minimum inhibitory concentration of the peptide against Salmonella enteritidis was found to be 150 nM. Evaluation of the solution conformation of the peptide using circular dichroism spectroscopy showed that the peptide is well folded in 40% trifluoroethanol with helical structure whereas the folded structure is lost in aqueous solution. To increase the yield of this potent peptide, we overexpressed GST-tagged microcin N using E. coli BL21. Recombinant GST-tagged microcin N was successfully expressed in E. coli BL21; however, the cleaved mature microcin N did not show activity against the indicator strain (S. enterica) most likely due to the extreme hydrophobic nature of the peptide. Efforts to produce active microcin N in large scale are discussed as this peptide has huge potential to be the next generation antimicrobial agent.
authors.affiliation: Chapman University School of Pharmacy (CUSP), Harry and Diane Rinker Health Science Campus, Chapman University, Irvine, CA 92618-1908, USA Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB T6G 2E1, Canada kkaur@chapman.edu.
authors.forename: Kamaljit
authors.initials: K
authors.lastname: Kaur
authors.affiliation: Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, Canada.
authors.forename: Oxana
authors.initials: O
authors.lastname: Tarassova
authors.affiliation: Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB T6G 2E1, Canada.
authors.forename: Ramana Venkata
authors.initials: RV
authors.lastname: Dangeti
authors.affiliation: Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB T6G 2E1, Canada.
authors.forename: Sarfuddin
authors.initials: S
authors.lastname: Azmi
authors.affiliation: Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, Canada.
authors.forename: David
authors.initials: D
authors.lastname: Wishart
authors.affiliation: Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB T6G 2P5, Canada.
authors.forename: Lynn
authors.initials: L
authors.lastname: McMullen
authors.affiliation: CanBiocin Inc., Edmonton, AB T5J 4P6, Canada.
authors.forename: Michael
authors.initials: M
authors.lastname: Stiles
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D001430:Bacteriocins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry; D008826:Microbial Sensitivity Tests; D009943:Organophosphorus Compounds; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry; D012477:Salmonella enteritidis / Q000187:drug effects; D017422:Sequence Analysis, DNA; D014270:Trifluoroethanol / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism; D056689:Uropathogenic Escherichia coli / Q000737:chemistry*
chemical_list: C043517:2-(4-isothiocyanatophenoxy)-1,3,2-dioxaphosphinene 2-oxide; D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D001430:Bacteriocins; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; C020411:microcin; D014270:Trifluoroethanol; D014357:Trypsin
keywords: antibacterial activity; bacteriocin; mass spectrometry: N-terminal sequencing; microcin N; recombinant microcin N