RESUMO
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.
Assuntos
Escherichia coli/genética , Expressão Gênica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Acetatos/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canais de Translocação SEC , Proteínas SecARESUMO
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.
Assuntos
Ácido Desoxicólico/química , Proteínas de Escherichia coli/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Cristalografia por Raios X , Ácido Desoxicólico/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismoRESUMO
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.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/citologia , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Cinética , Óperon Lac/genética , Proteínas de Fusão de Membrana/biossíntese , Proteínas de Fusão de Membrana/genética , Proteínas de Fusão de Membrana/metabolismo , Proteínas de Membrana Transportadoras/biossíntese , Proteínas de Membrana Transportadoras/metabolismo , Regiões Promotoras Genéticas/genética , Proteoma/metabolismo , Proteínas Virais/metabolismoRESUMO
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.