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Into the Membrane and Beyond: Improving Membrane Protein Over-Expression in Escherichia coli
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Jan-Willem de Gier)
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 [en]
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: urn:nbn:se:su:diva-57971ISBN: 978-91-7447-295-0 (print)OAI: oai:DiVA.org:su-57971DiVA: diva2:419025
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
List of papers
1. Immobilization of the first dimension in 2D blue native/SDS-PAGE allows the relative quantification of membrane proteomes
Open this publication in new window or tab >>Immobilization of the first dimension in 2D blue native/SDS-PAGE allows the relative quantification of membrane proteomes
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2008 (English)In: Methods, ISSN 1095-9130, Vol. 46, no 2, 48-53 p.Article in journal (Refereed) Published
Abstract [en]

In biological membranes many proteins are organized in complexes. The method of choice for the global analysis of the subunits of these complexes is two-dimensional blue native (2D BN)/SDS–PAGE. In the 1st dimension complexes are separated by BN-PAGE, and in the 2nd dimension their subunits are resolved by SDS–PAGE. In the currently available protocols the 1st dimension BN gel lanes get distorted during their transfer to the 2nd dimension separation gels. This leads to low reproducibility and high variation of 2D BN/SDS-gels, rendering them unsuitable for comparative analysis. We have developed a 2D BN/SDS–PAGE protocol where the 1st dimension BN gel is cast on a GelBond PAG film. Immobilization prevents distortion of BN gel lanes, which lowers variation and greatly improves reproducibility of 2D BN/SDS-gels. 2D BN/SDS–PAGE with an immobilized 1st dimension was used for the comparative analysis of the cytoplasmic membrane proteomes of Escherichia coli cells overexpressing a membrane protein and to create a 2D BN/SDS–PAGE reference map of the E. coli cytoplasmic membrane proteome with 143 identified proteins from 165 different protein spots.

Keyword
rane protein, Protein complex, Two-dimensional blue native SDS–PAGE, Comparative proteomics, E. coli
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-17270 (URN)10.1016/j.ymeth.2008.06.017 (DOI)000261367600002 ()18674622 (PubMedID)
Available from: 2009-01-12 Created: 2009-01-12 Last updated: 2011-06-15Bibliographically approved
2. Consequences of the overexpression of a eukaryotic membrane protein, the human KDEL receptor, in Escherichia coli
Open this publication in new window or tab >>Consequences of the overexpression of a eukaryotic membrane protein, the human KDEL receptor, in Escherichia coli
2011 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 407, no 4, 532-542 p.Article in journal (Refereed) Published
Abstract [en]

Escherichia coli is the most widely used host for producing membrane proteins. Thus far, to study the consequences of membrane protein overexpression in E. coli, we have focussed on prokaryotic membrane proteins as overexpression targets. Their overexpression results in the saturation of the Sec translocon, which is a protein-conducting channel in the cytoplasmic membrane that mediates both protein translocation and insertion. Saturation of the Sec translocon leads to (i) protein misfolding/aggregation in the cytoplasm, (ii) impaired respiration, and (iii) activation of the Arc response, which leads to inefficient ATP production and the formation of acetate. The overexpression yields of eukaryotic membrane proteins in E. coli are usually much lower than those of prokaryotic ones. This may be due to differences between the consequences of the overexpression of prokaryotic and eukaryotic membrane proteins in E. coli. Therefore, we have now also studied in detail how the overexpression of a eukaryotic membrane protein, the human KDEL receptor, affects E. coli. Surprisingly, the consequences of the overexpression of a prokaryotic and a eukaryotic membrane protein are very similar. Strain engineering and likely also protein engineering can be used to remedy the saturation of the Sec translocon upon overexpression of both prokaryotic and eukaryotic membrane proteins in E. coli.

Keyword
protein production, Sec translocon, membrane protein biogenesis, protein expression optimisation, proteomics
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-57976 (URN)10.1016/j.jmb.2011.02.007 (DOI)000288925200005 ()
Available from: 2011-05-25 Created: 2011-05-25 Last updated: 2017-12-11Bibliographically approved
3. 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: 2017-12-13Bibliographically approved
4. Evolution of theC41(DE3) and C43(DE3) strains
Open this publication in new window or tab >>Evolution of theC41(DE3) and C43(DE3) strains
(English)Manuscript (preprint) (Other academic)
National Category
Chemical Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-57985 (URN)
Available from: 2011-05-25 Created: 2011-05-25 Last updated: 2011-06-15Bibliographically approved
5. Escherichia coli peptide binding protein OppA has a preference for positively chargedpeptides
Open this publication in new window or tab >>Escherichia coli peptide binding protein OppA has a preference for positively chargedpeptides
(English)Manuscript (preprint) (Other academic)
National Category
Other Basic Medicine
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-57986 (URN)
Available from: 2011-05-25 Created: 2011-05-25 Last updated: 2011-06-15Bibliographically approved

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