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Consequences of the overexpression of a eukaryotic membrane protein, the human KDEL receptor, in Escherichia coli
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Mathematics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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.

Place, publisher, year, edition, pages
2011. Vol. 407, no 4, 532-542 p.
Keyword [en]
protein production, Sec translocon, membrane protein biogenesis, protein expression optimisation, proteomics
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-57976DOI: 10.1016/j.jmb.2011.02.007ISI: 000288925200005OAI: oai:DiVA.org:su-57976DiVA: diva2:419062
Available from: 2011-05-25 Created: 2011-05-25 Last updated: 2012-01-18Bibliographically approved
In thesis
1. 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

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