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Intracellular vesicles induced by monotopic membrane protein in Escherichia coli
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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 [en]
Membrane protein, intracellular vesicles, Escherichia coli, glycosyltransferase, overexpression, optimization, detergent, screening, lipid composition
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-29070ISBN: 978-91-7155-864-0 (print)OAI: oai:DiVA.org:su-29070DiVA: diva2:229034
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
List of papers
1. High-yield expression and purification of a monotopic membrane glycosyltransferase
Open this publication in new window or tab >>High-yield expression and purification of a monotopic membrane glycosyltransferase
2009 (English)In: Protein Expression and Purification, ISSN 1046-5928, 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
Keyword
Membrane protein, Monotopic, Overexpression, Optimization, Size-exclusion chromatography, Glycosyltransferase, Escherichia coli
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-29063 (URN)10.1016/j.pep.2009.03.008 (DOI)000266394800004 ()
Available from: 2009-08-10 Created: 2009-08-10 Last updated: 2009-08-11Bibliographically approved
2. Massive formation of intracellular membrane vesicles in Escherichia coli by a monotopic membrane-bound lipid glycosyltransferase
Open this publication in new window or tab >>Massive formation of intracellular membrane vesicles in Escherichia coli by a monotopic membrane-bound lipid glycosyltransferase
Show others...
2009 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 284, no 49, 33904-33914 p.Article in journal (Refereed) Published
Abstract [en]

The morphology and curvature of biological bilayers are determined by the packing shapes and interactions of their participant molecules. Bacteria, except photosynthetic groups, usually lack intracellular membrane organelles. Strong overexpression in Escherichia coli of a foreign monotopic glycosyltransferase (named monoglycosyldiacylglycerol synthase), synthesizing a nonbilayer-prone glucolipid, induced massive formation of membrane vesicles in the cytoplasm. Vesicle assemblies were visualized in cytoplasmic zones by fluorescence microscopy. These have a very low buoyant density, substantially different from inner membranes, with a lipid content of > or = 60% (w/w). Cryo-transmission electron microscopy revealed cells to be filled with membrane vesicles of various sizes and shapes, which when released were mostly spherical (diameter approximately 100 nm). The protein repertoire was similar in vesicle and inner membranes and dominated by the glycosyltransferase. Membrane polar lipid composition was similar too, including the foreign glucolipid. A related glycosyltransferase and an inactive monoglycosyldiacylglycerol synthase mutant also yielded membrane vesicles, but without glucolipid synthesis, strongly indicating that vesiculation is induced by the protein itself. The high capacity for membrane vesicle formation seems inherent in the glycosyltransferase structure, and it depends on the following: (i) lateral expansion of the inner monolayer by interface binding of many molecules; (ii) membrane expansion through stimulation of phospholipid synthesis, by electrostatic binding and sequestration of anionic lipids; (iii) bilayer bending by the packing shape of excess nonbilayer-prone phospholipid or glucolipid; and (iv) potentially also the shape or penetration profile of the glycosyltransferase binding surface. These features seem to apply to several other proteins able to achieve an analogous membrane expansion.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-34581 (URN)10.1074/jbc.M109.021618 (DOI)000272165200023 ()19767390 (PubMedID)
Available from: 2010-01-11 Created: 2010-01-11 Last updated: 2017-12-12Bibliographically approved
3. Increased amounts of overexpressed membrane proteins in Escherichia coli by co-expression with a foreign vesicle-inducing protein
Open this publication in new window or tab >>Increased amounts of overexpressed membrane proteins in Escherichia coli by co-expression with a foreign vesicle-inducing protein
(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.

Identifiers
urn:nbn:se:su:diva-29069 (URN)
Available from: 2009-08-10 Created: 2009-08-10 Last updated: 2010-01-14Bibliographically approved
4. Lipid-engineered Escherichia coli membranes reveal critical lipid headgroup size for protein function.
Open this publication in new window or tab >>Lipid-engineered Escherichia coli membranes reveal critical lipid headgroup size for protein function.
Show others...
2009 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 284, no 2, 954-65 p.Article in journal (Refereed) Published
Abstract [en]

Escherichia coli membranes have a substantial bilayer curvature stress due to a large fraction of the nonbilayer-prone lipid phosphatidylethanolamine, and a mutant (AD93) lacking this lipid is severely crippled in several membrane-associated processes. Introduction of four lipid glycosyltransferases from Acholeplasma laidlawii and Arabidopsis thaliana, synthesizing large amounts of two nonbilayer-prone, and two bilayer-forming gluco- and galacto-lipids, (i) restored the curvature stress with the two nonbilayer lipids, and (ii) diluted the high negative lipid surface charge in all AD93 bilayers. Surprisingly, the bilayer-forming diglucosyl-diacylglycerol was almost as good in improving AD93 membrane processes as the two nonbilayer-prone glucosyl-diacylglycerol and galactosyl-diacylglycerol lipids, strongly suggesting that lipid surface charge dilution by these neutral lipids is very important for E. coli. Increased acyl chain length and unsaturation, plus cardiolipin (nonbilayer-prone) content, were probably also beneficial in the modified strains. However, despite a correct transmembrane topology for the transporter LacY in the diglucosyl-diacylglycerol clone, active transport failed in the absence of a nonbilayer-prone glycolipid. The corresponding digalactosyl-diacylglycerol bilayer lipid did not restore AD93 membrane processes, despite analogous acyl chain and cardiolipin contents. Chain ordering, probed by bis-pyrene lipids, was substantially lower in the digalactosyl-diacylglycerol strain lipids due to its extended headgroup. Hence, a low surface charge density of anionic lipids is important in E. coli membranes, but is inefficient if the headgroup of the diluting lipid is too large. This strongly indicates that a certain magnitude of the curvature stress is crucial for the bilayer in vivo.

Identifiers
urn:nbn:se:su:diva-29065 (URN)10.1074/jbc.M804482200 (DOI)000262122900031 ()18981182 (PubMedID)
Available from: 2009-08-10 Created: 2009-08-10 Last updated: 2009-08-11Bibliographically approved

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