Dataset Information

Improving the purification of membrane proteins from Escherichia coli C41(DE3)

ABSTRACT: The overexpression and purification of membrane proteins to high purity and homogeneity is a challenging task. Over time, several strains have been developed that decrease the toxic side-effects and thus result in higher bio mass and protein yield. However, two major contaminants have been identified in membrane protein preparations from E. coli: the outer membrane porin OmpF and AcrB, which is part of a tripartite efflux pump. Both proteins crystallise from low concentrations and diverse conditions, which make them a major problem, especially in membrane protein crystallography. In this study, we present a C41(DE3)-derived expression strains that is depleted of these two proteins.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Escherichia Coli

SUBMITTER: Gereon Poschmann

LAB HEAD: Gereon Poschmann

PROVIDER: PXD011437 | Pride | 2019-11-12

REPOSITORIES: Pride

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Publications

Shaping the lipid composition of bacterial membranes for membrane protein production.

Kanonenberg Kerstin K Royes Jorge J Kedrov Alexej A Poschmann Gereon G Angius Federica F Solgadi Audrey A Spitz Olivia O Kleinschrodt Diana D Stühler Kai K Miroux Bruno B Schmitt Lutz L

Microbial cell factories 20190810 1

<h4>Background</h4>The overexpression and purification of membrane proteins is a bottleneck in biotechnology and structural biology. E. coli remains the host of choice for membrane protein production. To date, most of the efforts have focused on genetically tuning of expression systems and shaping membrane composition to improve membrane protein production remained largely unexplored.<h4>Results</h4>In E. coli C41(DE3) strain, we deleted two transporters involved in fatty acid metabolism (OmpF a ...[more]

PMID: 31400768

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Project description:Transcriptome profiles were analyzed using the samples taken at the exponential and stationary phases during the cultivation of REL606 and MG1655 in LB medium. At the exponential growth phase, most highly expressed genes of B were those for replication, translation, or nucleotide transport and metabolism, while many of the K-12 genes were involved in cell motility, transcription, carbohydrate transport, or energy production. At the stationary phase, many of the genes highly expressed in B were for transport and metabolism of various amino acids and carbohydrates, whereas those in K-12 had functions related to cell motility, ribosomal subunit protein production, or energy generation. Many genes in REL606 and MG1655 showed highly distinct expression levels irrespective of growth conditions. Highly expressed genes in REL606 included those encoding enzymes for biosynthesis of L-arginine (argAGDECBHI) and branched-chain amino acid (ilvGMEDA), and those encoding a subunit of the L-arginine transporter (artJ), cytochrome b562 (cybC), subunits of the histidine ABC transporter (hisPJ), cytotoxins (hokED), outer membrane porin (ompF), L-arginine decarboxylase (speA), and cell division inhibitor (sulA). Highly expressed genes in MG1655 included those for chemotaxis (cheZYRWA, tap, trg, tsr), Lon protease (lon), C4-dicarboxylate-sensing histidine kinase (dcuS), chaperones (clpB, dnaK, groES, htpG, ibpA), the major subunit of type 1 fimbriae (fimA), a regulator of flagellar biosynthesis (flhC), glycerol-3-phosphate-dehydrogenase (glpABCD), glycerophosphoryl diester phosphodiesterase (glpQ), glycerol-3-phosphate transporter (glpT), hydrogenase 2 (hybCBO), outer membrane porins (nmpC, ompA, ompC), and galactitol transport and metabolism (gatYZC). All microarray experiments were performed in duplicate with dye-swapping. The probes were spotted in duplicate onto the microarray. Thus, the log2 of the intensity ratio with the two dyes for each spot was calculated from up to four values from the duplicated spot on the microarray and the dye-swap experiment.

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Dataset Information

Improving the purification of membrane proteins from Escherichia coli C41(DE3)

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Publications

Shaping the lipid composition of bacterial membranes for membrane protein production.

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