Isolation and characterization of the outer membrane of Escherichia coli with autodisplayed Z-domains

• Published in: Biochimica et biophysica acta Vol. 1848; no. 3; pp. 842 - 847
• Main Authors: Park, Min, Yoo, Gu, Bong, Ji-Hong, Jose, Joachim, Kang, Min-Jung, Pyun, Jae-Chul
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-03-2015
• Subjects: autodisplay; Bacterial Outer Membrane Proteins - chemistry; Bacterial Outer Membrane Proteins - metabolism; beta-Galactosidase - chemistry; beta-Galactosidase - metabolism; beta-Lactamases - chemistry; beta-Lactamases - metabolism; Blotting, Western; Cell Membrane - chemistry; Cell Membrane - metabolism; E. coli; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; Lipid Bilayers - chemistry; Lipid Bilayers - metabolism; Models, Molecular; Multienzyme Complexes - chemistry; Multienzyme Complexes - metabolism; NADH, NADPH Oxidoreductases - chemistry; NADH, NADPH Oxidoreductases - metabolism; outer membrane; Protein Structure, Tertiary; Z-domain; outer membrane; autodisplay; Z-domain; E. coli
• ISSN: 0005-2736; 0006-3002; 1879-2642
• DOI: 10.1016/j.bbamem.2014.12.011

“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.
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•Z-domains were autodisplayed on the outer membrane (OM) of E. coli.•The OM of E. coli with autodisplayed Z-domains was isolated into liposomes.•The purity of the isolated OM was analyzed by Western blots of marker proteins.•Isolated OM was assayed for marker enzymes from each part of E. coli.•Yield of OM isolation was determined to be 80% with less than 1% contaminants.