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High-yield expression and purification of a monotopic membrane glycosyltransferase
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. (Åke Wieslander)
Umeå Universitet.
Massachusetts Institute of Technology.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2009 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 66, no 2, 143-148 p.Article in journal (Refereed) Published
Abstract [en]

Membrane proteins are essential to many cellular processes. However, the systematic study of membrane protein structure has been hindered by the difficulty in obtaining large quantities of these proteins. Protein overexpression using Escherichia coli is commonly used to produce large quantities of protein, but usually yields very little membrane protein. Furthermore, optimization of the expressing conditions, as well as the choice of detergent and other buffer components, is thought to be crucial for increasing the yield of stable and homogeneous protein. Herein we report high-yield expression and purification of a membrane-associated monotopic protein, the glycosyltransferase monoglucosyldiacylglycerol synthase (alMGS), in E. coli. Systematic optimization of protein expression was achieved through controlling a few basic expression parameters, including temperature and growth media, and the purifications were monitored using a fast and efficient size-exclusion chromatography (SEC) screening method. The latter method was shown to be a powerful tool for fast screening and for finding the optimal protein-stabilizing conditions. For alMGS it was found that the concentration of detergent was just as important as the type of detergent, and a low concentration of n-Dodecyl-β-D-maltoside (DDM) (~1× critical micelle concentration) was the best for keeping the protein stable and homogeneous. By using these simply methods to optimize the conditions for alMGS expression and purification, the final expression level increase by two orders of magnitude, reaching 170 mg of pure protein per litre culture.

Place, publisher, year, edition, pages
Elsevier , 2009. Vol. 66, no 2, 143-148 p.
Keyword [en]
Membrane protein, Monotopic, Overexpression, Optimization, Size-exclusion chromatography, Glycosyltransferase, Escherichia coli
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-29063DOI: 10.1016/j.pep.2009.03.008ISI: 000266394800004OAI: oai:DiVA.org:su-29063DiVA: diva2:229026
Available from: 2009-08-10 Created: 2009-08-10 Last updated: 2017-12-13Bibliographically 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.
Keyword
Membrane protein, intracellular vesicles, Escherichia coli, glycosyltransferase, overexpression, optimization, detergent, screening, lipid composition
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
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)
Opponent
Supervisors
Note
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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