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1.
J Mol Biol ; 407(4): 532-42, 2011 Apr 08.
Article in English | MEDLINE | ID: mdl-21316372

ABSTRACT

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.


Subject(s)
Escherichia coli/genetics , Gene Expression , Membrane Proteins/genetics , Membrane Proteins/metabolism , Receptors, Peptide/genetics , Receptors, Peptide/metabolism , Acetates/metabolism , Adenosine Triphosphatases/metabolism , Bacterial Proteins/metabolism , Humans , Membrane Transport Proteins/metabolism , Protein Engineering , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SEC Translocation Channels , SecA Proteins
2.
Mol Membr Biol ; 25(8): 677-82, 2008 Dec.
Article in English | MEDLINE | ID: mdl-19023693

ABSTRACT

Gastrointestinal bacteria, like Escherichia coli, must remove bile acid to survive in the gut. Bile acid removal in E. coli is thought to be mediated primarily by the multidrug efflux pump, AcrB. Here, we present the structure of E. coli AcrB in complex with deoxycholate at 3.85 A resolution. All evidence suggests that bile acid is transported out of the cell via the periplasmic vestibule of the AcrAB-TolC complex.


Subject(s)
Deoxycholic Acid/chemistry , Escherichia coli Proteins/chemistry , Multidrug Resistance-Associated Proteins/chemistry , Crystallography, X-Ray , Deoxycholic Acid/metabolism , Escherichia coli/chemistry , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Multidrug Resistance-Associated Proteins/metabolism , Mutant Proteins/chemistry , Mutant Proteins/metabolism
3.
Proc Natl Acad Sci U S A ; 105(38): 14371-6, 2008 Sep 23.
Article in English | MEDLINE | ID: mdl-18796603

ABSTRACT

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.


Subject(s)
Escherichia coli Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Membrane Transport Proteins/genetics , DNA-Directed RNA Polymerases/metabolism , Escherichia coli/cytology , Escherichia coli/growth & development , Escherichia coli Proteins/biosynthesis , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial , Kinetics , Lac Operon/genetics , Membrane Fusion Proteins/biosynthesis , Membrane Fusion Proteins/genetics , Membrane Fusion Proteins/metabolism , Membrane Transport Proteins/biosynthesis , Membrane Transport Proteins/metabolism , Promoter Regions, Genetic/genetics , Proteome/metabolism , Viral Proteins/metabolism
4.
J Biol Chem ; 283(26): 17881-90, 2008 Jun 27.
Article in English | MEDLINE | ID: mdl-18456666

ABSTRACT

The polytopic inner membrane protein MalF is a constituent of the MalFGK(2) maltose transport complex in Escherichia coli. We have studied the biogenesis of MalF using a combination of in vivo and in vitro approaches. MalF is targeted via the SRP pathway to the Sec/YidC insertion site. Despite close proximity of nascent MalF to YidC during insertion, YidC is not required for the insertion of MalF into the membrane. However, YidC is required for the stability of MalF and the formation of the MalFGK(2) maltose transport complex. Our data indicate that YidC supports the folding of MalF into a stable conformation before it is incorporated into the maltose transport complex.


Subject(s)
ATP-Binding Cassette Transporters/physiology , Escherichia coli Proteins/physiology , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial , Maltose/metabolism , Membrane Transport Proteins/physiology , Monosaccharide Transport Proteins/physiology , ATP-Binding Cassette Transporters/metabolism , Biological Transport , Cell Membrane/metabolism , Escherichia coli Proteins/metabolism , Membrane Transport Proteins/metabolism , Models, Biological , Monosaccharide Transport Proteins/metabolism , Plasmids/metabolism , Protein Binding , Protein Conformation , Protein Folding
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