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Consequences of membrane protein overexpression in Escherichia coli.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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2007 (English)In: Mol Cell Proteomics, ISSN 1535-9476, Vol. 6, no 9, 1527-50 p.Article in journal (Other academic) Published
Place, publisher, year, edition, pages
2007. Vol. 6, no 9, 1527-50 p.
Keyword [en]
Adenosine Triphosphate/chemistry, Cell Membrane/metabolism, Computational Biology/methods, Cytoplasm/metabolism, Electrophoresis; Gel; Two-Dimensional, Escherichia coli/*metabolism, Escherichia coli Proteins/*chemistry, Green Fluorescent Proteins/metabolism, Hydrogen-Ion Concentration, Mass Spectrometry/methods, Membrane Proteins/*chemistry, Microscopy; Fluorescence, Plasmids/metabolism, Protein Transport, Proteomics/methods
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:su:diva-21326ISI: 000249237200006PubMedID: 17446557OAI: oai:DiVA.org:su-21326DiVA: diva2:187852
Available from: 2008-01-14 Created: 2008-01-14 Last updated: 2011-01-11Bibliographically approved
In thesis
1. From Biogenesis to Overexpression of Membrane Proteins in Escherichia coli
Open this publication in new window or tab >>From Biogenesis to Overexpression of Membrane Proteins in Escherichia coli
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In both pro- and eukaryotes 20-30% of all genes encode alpha-helical transmembrane domain proteins, which act in various and often essential capacities. Notably, membrane proteins play key roles in disease and they constitute more than half of all known drug targets.

The natural abundance of membrane proteins is in general too low to conveniently isolate sufficient material for functional and structural studies. Therefore, most membrane proteins have to be obtained through overexpression. Escherichia coli is one of the most successful hosts for overexpression of recombinant proteins. While the production of soluble proteins is comparably straightforward, overexpression of membrane proteins remains a challenging task. The yield of membrane localized recombinant membrane protein is usually low and inclusion body formation is a serious problem. Furthermore, membrane protein overexpression is often toxic to the host cell. Although several reasons can be postulated, the basis of these difficulties is not completely understood, preventing the design of rational strategies to improve membrane protein overexpression yields.

The objective of my Ph.D. studies has been to improve membrane protein overexpression in E. coli by a) understanding membrane protein overexpression from the perspective of membrane protein biogenesis, b) systematically investigating the physiological response to overexpression of membrane proteins and c) engineering strains that are optimized for membrane protein overexpression based on insights resulting from these studies.

By working toward these objectives, I was able to identify and alleviate one of the major bottlenecks of membrane protein overexpression in E. coli: saturation of the Sec-translocon could be overcome by harmonizing translation and membrane insertion of the recombinant membrane protein. This minimized the toxic effects of overexpression and thus resulted in increased membrane protein-producing biomass.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik, 2008. 91 p.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-7513 (URN)978-91-7155-594-6 (ISBN)
Public defence
2008-05-23, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00 (English)
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Available from: 2008-04-30 Created: 2008-04-30 Last updated: 2010-01-13Bibliographically approved

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