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From Biogenesis to Overexpression of Membrane Proteins in Escherichia coli
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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: urn:nbn:se:su:diva-7513ISBN: 978-91-7155-594-6 (print)OAI: oai:DiVA.org:su-7513DiVA: diva2:198474
Public defence
2008-05-23, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2008-04-30 Created: 2008-04-30 Last updated: 2010-01-13Bibliographically approved
List of papers
1. Effects of SecE depletion on the inner and outer membrane proteomes of Escherichia coli
Open this publication in new window or tab >>Effects of SecE depletion on the inner and outer membrane proteomes of Escherichia coli
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2008 (English)In: Journal of Bacteriology, ISSN 0021-9193, Vol. 190, no 10, 3505-3525 p.Article in journal (Refereed) Published
Keyword
Bacterial Outer Membrane Proteins/metabolism, Cell Membrane/*metabolism, Escherichia coli/*genetics/metabolism, Escherichia coli Proteins/chemistry/*metabolism, Mass Spectrometry, Membrane Transport Proteins/chemistry/metabolism/*physiology, Proteome/*metabolism, Proteomics/*methods
Identifiers
urn:nbn:se:su:diva-16865 (URN)10.1128/JB.01631-07 (DOI)000255622500011 ()18296516 (PubMedID)
Available from: 2008-12-29 Created: 2008-12-29 Last updated: 2010-01-27Bibliographically approved
2. Biogenesis of MalF and the MalFGK2 maltose transport complex in Escherichia coli requires YidC
Open this publication in new window or tab >>Biogenesis of MalF and the MalFGK2 maltose transport complex in Escherichia coli requires YidC
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2008 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, no 283, 17881-17890 p.Article in journal (Refereed) Published
Abstract [en]

The polytopic inner membrane protein MalF is a constituent of the MalFGK2 maltose transport complex in Escherichia coli. We have studied the biogenesis of MalF using a combination of in vivo and in vitro approaches. MalF is targeted via the SRP pathway to the Sec/YidC insertion site. Despite close proximity of nascent MalF to YidC during insertion, YidC is not required for the insertion of MalF into the membrane. However, YidC is required for the stability of MalF and the formation of the MalFGK2 maltose transport complex. Our data indicate that YidC supports the folding of MalF into a stable conformation before it is incorporated into the maltose transport complex.

Keyword
Membrane Transport, Structure, Function, and Biogenesis
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-24886 (URN)10.1074/jbc.M801481200 (DOI)
Available from: 2008-04-30 Created: 2008-04-30 Last updated: 2011-03-16Bibliographically approved
3. Consequences of membrane protein overexpression in Escherichia coli.
Open this publication in new window or tab >>Consequences of membrane protein overexpression in Escherichia coli.
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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
Keyword
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:nbn:se:su:diva-21326 (URN)000249237200006 ()17446557 (PubMedID)
Available from: 2008-01-14 Created: 2008-01-14 Last updated: 2011-01-11Bibliographically approved
4. Tuning Escherichia coli for membrane protein overexpression
Open this publication in new window or tab >>Tuning Escherichia coli for membrane protein overexpression
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2008 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 38, 14371-17376 p.Article in journal (Refereed) Published
Abstract [en]

A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used “Walker strains” C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.

Keyword
engineering, systems biotechnology, proteomics
National Category
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-24888 (URN)10.1073/pnas.0804090105 (DOI)000259592400028 ()
Available from: 2008-04-30 Created: 2008-04-30 Last updated: 2013-10-21Bibliographically approved

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