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

Min Park; Gu Yoo; Ji Hong Bong; Joachim Jose; Min Jung Kang; Jae Chul Pyun

doi:10.1016/j.bbamem.2014.12.011

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

Min Park, Gu Yoo, Ji Hong Bong, Joachim Jose, Min Jung Kang, Jae Chul Pyun

Department of Materials Science and Engineering

Research output: Contribution to journal › Article › peer-review

43 Citations (Scopus)

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.

Original language	English
Pages (from-to)	842-847
Number of pages	6
Journal	Biochimica et Biophysica Acta - Biomembranes
Volume	1848
Issue number	3
DOIs	https://doi.org/10.1016/j.bbamem.2014.12.011
Publication status	Published - 2015 Jan 15

Bibliographical note

Funding Information:
This research was supported by the National Research Foundation of Korea ( 2013-056267 , 2013-056331 ). This work was also supported by the Creative Fusion Research Program through the Creative Allied Project funded by Korea Research Council of Fundamental Science and Technology ( CAP-12-1 ).

Publisher Copyright:
© 2014 Elsevier B.V.

All Science Journal Classification (ASJC) codes

Biophysics
Biochemistry
Cell Biology

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10.1016/j.bbamem.2014.12.011

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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.",

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AU - Jose, Joachim

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AU - Pyun, Jae Chul

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

Abstract

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