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Tuning Escherichia coli for membrane protein overexpression
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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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.

Place, publisher, year, edition, pages
2008. Vol. 105, no 38, 14371-17376 p.
Keyword [en]
engineering, systems biotechnology, proteomics
National Category
Organic Chemistry
Identifiers
URN: urn:nbn:se:su:diva-24888DOI: 10.1073/pnas.0804090105ISI: 000259592400028OAI: oai:DiVA.org:su-24888DiVA: diva2:198473
Available from: 2008-04-30 Created: 2008-04-30 Last updated: 2017-12-13Bibliographically 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)
Opponent
Supervisors
Available from: 2008-04-30 Created: 2008-04-30 Last updated: 2010-01-13Bibliographically approved
2. Into the Membrane and Beyond: Improving Membrane Protein Over-Expression in Escherichia coli
Open this publication in new window or tab >>Into the Membrane and Beyond: Improving Membrane Protein Over-Expression in Escherichia coli
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Membrane proteins fulfil a wide variety of essential functions in the cell and many are (potential) drug targets. Since their natural abundance is usually very low, most membrane proteins have to be over-expressed for functional and structural studies. T7 RNA polymerase (T7RNAP) based Escherichia coli strains, like BL21(DE3), are very popular protein production hosts. Unfortunately, over-expression of membrane proteins in E. coli is usually toxic to the cells. During my Ph.D. I have tried to understand the reasons for this toxicity by studying the consequences of membrane protein over-expression using a combination of proteomics and more focused biochemical and genetic methods. First, we had to improve the existing 2D BN/SDS-PAGE protocol to perform reliable comparative analysis of membrane proteomes. With the new protocol I have studied the effects of the expression of membrane proteins, including the human KDEL receptor, on BL21(DE3) and its derivatives, C41(DE3) and C43(DE3) (a.k.a. the Walker strains). The latter two were isolated for their improved membrane protein over-expression characteristics. Saturation of the Sec translocon, a cytoplasmic membrane associated protein conducting channel that mediates the insertion/biogenesis of membrane proteins, appeared to be the prime reason for the toxicity of membrane protein over-expression. Therefore, it was not surprising that we have identified mutations in the promoter governing the expression of the T7RNAP in the C41(DE3) and C43(DE3) strains that weaken it compared to the one in BL21(DE3). Based on this observation, we have engineered a plasmid (pLemo) with the gene encoding the natural inhibitor of T7RNAP, T7 lysozyme, under the control of the titratable rhamnose promoter. With the help of this plasmid the activity of the T7RNAP can be precisely set thereby avoiding saturation of the Sec translocon upon membrane protein over-expression. However, we have identified more changes in the Walker strains. Notable examples are the up regulation of peptide transporters in C41(DE3) and the expression of the Lon protease in C43(DE3). To study peptide import in E. coli I have characterized the in C41(DE3) strongly up regulated periplasmic binding protein OppA using a combination of biochemical and structural methods. The obtained data have resulted in many leads and ideas to further improve membrane protein over-expression yields in E. coli.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2011. 79 p.
Keyword
Escherichia coli, membrane protein over-expression, proteomics, T7 RNA polymerase, peptide transport, strain engineering
National Category
Biochemistry and Molecular Biology Physical Chemistry
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-57971 (URN)978-91-7447-295-0 (ISBN)
Public defence
2011-09-02, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.Available from: 2011-08-11 Created: 2011-05-25 Last updated: 2011-06-15Bibliographically approved
3. From protein production to genome evolution in Escherichia coli
Open this publication in new window or tab >>From protein production to genome evolution in Escherichia coli
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The aim of my Ph.D. studies was to improve production yields of membrane- and secretory proteins in the widely used E. coli protein production strain BL21(DE3). In this strain expression of the gene encoding the protein of interest is driven by the powerful T7 RNA polymerase (T7 RNAP) whose gene is located on the chromosome and under control of the strong, IPTG-inducible lacUV5 promoter. Unfortunately, the production of many membrane and secretory proteins is 'toxic' to BL21(DE3), resulting in poor growth and low production yields.

To understand this ‘toxicity’, the BL21(DE3) derived mutant strains C41(DE3) and C43(DE3) were characterized. Somehow, these strains can efficiently produce many ‘toxic’ membrane and secretory proteins. We showed that mutations weakening the lacUV5 promoter are responsible for this. These mutations result in a slower onset of protein production upon the addition of IPTG, which avoids saturating the Sec-translocon capacity. The Sec-translocon is a protein-conducting channel in the cytoplasmic membrane mediating the biogenesis of membrane proteins and translocation of secretory proteins. Next, we constructed a BL21(DE3)-derivative, Lemo21(DE3), in which the activity of T7 RNAP can be precisely controlled by titrating in its natural inhibitor T7 lysozyme using the rhamnose promoter system. In Lemo21(DE3), the expression level of genes encoding membrane and secretory proteins can be set such that the Sec-translocon capacity is not saturated. This is key to optimizing membrane and secretory protein production yields. Finally, reconstructing the evolution of C41(DE3) from BL21(DE3) in real time showed that during its isolation C41(DE3) had acquired mutations critical for surviving the starvation conditions used, and provided insight in how the mutations in the lacUV5 promoter had occurred.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2013. 59 p.
Keyword
Escherichia coli, BL21(DE3), protein production, membrane proteins, secretory proteins, genome evolution
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-94993 (URN)978-91-7447-786-3 (ISBN)
Public defence
2013-11-22, Magnelisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

Available from: 2013-10-30 Created: 2013-10-20 Last updated: 2013-10-29Bibliographically approved

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Klepsch, Mirjam M.Schlegel, SusanDraheim, RogerTarry, MichaelHögbom, MartinPersson, Jan O.de Gier, Jan-Willem
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