title: Isolation and characterization of the outer membrane of Escherichia coli with autodisplayed Z-domains.
abstract: "Autodisplay technology" is an expression technique used to display the various recombinant proteins on the outer membrane (OM) of Escherichia coli. The resulting autodisplayed Z-domain has been used to improve the sensitivity of immunoassays. In this work, a facile isolation method of the OM fraction of E. coli with autodisplayed Z-domains was presented using (1) an enzyme reaction for the hydrolysis of the peptidoglycan layer and (2) short centrifugation steps. The purity of the isolated OM fraction was analyzed. For the estimation of contamination with bacterial proteins from other parts of E. coli, Western blots of marker proteins for the OM (OmpA), periplasm (β-lactamase), inner membrane (SecA), and cytoplasm (β-galactosidase) were performed. Additionally, assays of marker components or enzymes from each part of E. coli were carried out including the OM (KDO), inner membrane (NADH oxidase), periplasm (β-lactamase), and cytoplasm (β-galactosidase). The yield of OM isolation using this new method was determined to be 80% of the total OM amount, with less than 1% being contaminants from other parts of E. coli.
authors.affiliation: Department of Materials Science and Engineering, Yonsei University, 50 Yonsei-Ro, Seodaemun-Gu, Seoul, 120-742, Korea; Korea Institute of Science and Technology (KIST), Seoul, Korea.
authors.forename: Min
authors.initials: M
authors.lastname: Park
authors.affiliation: Department of Materials Science and Engineering, Yonsei University, 50 Yonsei-Ro, Seodaemun-Gu, Seoul, 120-742, Korea.
authors.forename: Gu
authors.initials: G
authors.lastname: Yoo
authors.affiliation: Department of Materials Science and Engineering, Yonsei University, 50 Yonsei-Ro, Seodaemun-Gu, Seoul, 120-742, Korea.
authors.forename: Ji-Hong
authors.initials: JH
authors.lastname: Bong
authors.affiliation: Institute of Pharmaceutical and Medical Chemistry, University of Muenster, Muenster, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Jose
authors.affiliation: Korea Institute of Science and Technology (KIST), Seoul, Korea.
authors.forename: Min-Jung
authors.initials: MJ
authors.lastname: Kang
authors.affiliation: Department of Materials Science and Engineering, Yonsei University, 50 Yonsei-Ro, Seodaemun-Gu, Seoul, 120-742, Korea. Electronic address: jcpyun@yonsei.ac.kr.
authors.forename: Jae-Chul
authors.initials: JC
authors.lastname: Pyun
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D001616:beta-Galactosidase / Q000737:chemistry / Q000378:metabolism; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D009097:Multienzyme Complexes; C078167:OMPA outer membrane proteins; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D001616:beta-Galactosidase; D001618:beta-Lactamases
keywords: E. coli; Z-domain; autodisplay; outer membrane