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Increased amounts of overexpressed membrane proteins in Escherichia coli by co-expression with a foreign vesicle-inducing protein
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Escherichia coli has a limited capacity to overexpress integral membrane proteins to amounts needed for structural studies. This is usually attributed to the limited capacity of the Sec transport machinery, shortage of accessory chaperons, sub-optimal codon usage, potentially “wrong” lipids, and lack of membrane space for the new proteins. A foreign, monotopic lipid glycosyltransferase was recently shown to induce the formation of extensive amounts of intracellular vesicles in E. coli. We show here that such vesicles can improve the expressed levels up to 3-4 times for a substantial fraction of integral membrane proteins tested. These had 2 to 12 transmembrane helices, and all had a C-terminally fused GFP reporter. Strongly overexpressed proteins yielded intensely green vesicles, of slightly lower buoyant density than the inner membranes. Most proteins could be detected in the vesicles. Multivariate sequence analyses indicated a correlation between sequence property features and expression levels, and factors analyzed involved protein mass, transmembrane segments, inside/outside loops, etc. It is concluded that this vesicular system can yield substantial improvements in expression levels, by creation of extra membranes and lateral space in E. coli.

URN: urn:nbn:se:su:diva-29069OAI: diva2:229031
Available from: 2009-08-10 Created: 2009-08-10 Last updated: 2010-01-14Bibliographically approved
In thesis
1. Intracellular vesicles induced by monotopic membrane protein in Escherichia coli
Open this publication in new window or tab >>Intracellular vesicles induced by monotopic membrane protein in Escherichia coli
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The monotopic membrane protein alMGS, a glycosyltransferase catalyzing glucolipid synthesis in Acholeplasma laidlawii, was overexpressed in Escherichia coli. Optimization of basic growth parameters was performed, and a novel method for detergent and buffer screening using a small size-exclusion chromatography was developed. This resulted in a tremendous increase in protein yields, as well as the unexpected discovery that the protein induces intracellular vesicle formation in E. coli. This was confirmed by sucrose density separation and Cryo-TEM of membranes, and the properties of the vesicles were analyzed using SDS-PAGE, western blot and lipid composition analysis. It is concluded that both alMGS and alDGS, the next enzyme in glucolipid pathway, have the ability to make the membrane bend and eventually form vesicles. This is likely due to structural and electrostatic properties, such as the way the proteins penetrate the membrane interface and thereby expand one monolayer. The highly positively charged binding surfaces of the glycosyltransferases may bind negatively charged lipids, such as Phosphatidylglycerol (PG), in the membrane and withdraw it from the general pool of lipids. This would increase the overall lipid synthesis, since PG is a pace-keeper, and the local concentration of nonbilayer prone lipids, such as Phosphatidylethanolamine, can increase and also induce bending of the membrane. The formation of surplus membrane inside the E. coli cell was used to develop a generic method for overexpression of membrane proteins. A proof-of-principle experiment with a test set of twenty membrane proteins from E. coli resulted in elevated expression levels for about half of the set. Thus, we believe that this method will be a useful tool for overexpression of many membrane proteins. By engineering E. coli mutants with different lipid compositions, fine-tuning membrane properties for different proteins is also possible.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2009. 64 p.
Membrane protein, intracellular vesicles, Escherichia coli, glycosyltransferase, overexpression, optimization, detergent, screening, lipid composition
National Category
Biochemistry and Molecular Biology
Research subject
urn:nbn:se:su:diva-29070 (URN)978-91-7155-864-0 (ISBN)
Public defence
2009-09-18, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00 (English)
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 3: Manuscript.Available from: 2009-08-26 Created: 2009-08-10 Last updated: 2011-09-08Bibliographically approved

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Eriksson, Hanna M.Wieslander, Åke
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