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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(More)
Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft and thus prevent neurotoxicity. The proteins belong to a large and widespread family of secondary transporters, including bacterial glutamate, serine, and C4-dicarboxylate transporters; mammalian neutral-amino-acid transporters; and an(More)
Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft. The proteins belong to a large family of secondary transporters, which includes bacterial glutamate transporters. The C-terminal half of the glutamate transporters is well conserved and thought to contain the translocation path and the binding(More)
Escherichia coli BL21(DE3) is widely used to overexpress proteins. In this overexpression host, the gene encoding the target protein is located on a plasmid and is under control of the T7 promoter, which is recognized exclusively by the T7 RNA polymerase (RNAP). The T7 RNAP gene is localized on the chromosome, and its expression is governed by the(More)
The first biochemical and spectroscopic characterization of a purified membrane transporter for riboflavin (vitamin B(2)) is presented. The riboflavin transporter RibU from the bacterium Lactococcus lactis was overexpressed, solubilized, and purified. The purified transporter was bright yellow when the cells had been cultured in rich medium. We used a(More)
Transport of solutes between the cytosol and the vacuolar lumen is of crucial importance for various functions of vacuoles, including ion homeostasis; detoxification; storage of different molecules such as amino acids, phosphate, and calcium ions; and proteolysis. To identify proteins that catalyze solute transport across the vacuolar membrane, the membrane(More)
Energy-coupling factor (ECF) transporters belong to the ATP-binding cassette (ABC)-transporter family and mediate the uptake of essential micronutrients in many prokaryotic species. Two crystal structures of bacterial ECF transporters have recently been obtained and suggest that transport involves an unprecedented re-orientation of a membrane protein in the(More)
ATP-binding cassette (ABC) transporters form a large superfamily of ATP-dependent protein complexes that mediate transport of a vast array of substrates across membranes. The 14 currently available structures of ABC transporters have greatly advanced insight into the transport mechanism and revealed a tremendous structural diversity. Whereas the domains(More)
Many membrane proteins consist of bundles of alpha-helices that are reflected in typical hydropathy profiles of the amino acid sequences. The profiles provide a link between the amino acid sequence of the polypeptide chain and its folding and are much better conserved during evolution than the amino acid sequences from which they are deduced. In this paper,(More)
An affinity tag consisting of six adjacent histidine residues followed by an enterokinase cleavage site was genetically engineered at the N-terminus of the glutamate transport protein GltT of the thermophilic bacterium Bacillus stearothermophilus. The fusion protein was expressed in Escherichia coli and shown to transport glutamate. The highest levels of(More)