Therefore, in this study, we have purified a Strep-tagged derivative of E. coli FocA and demonstrated
that it is indeed an α-helical integral membrane protein. Surprisingly, however, FocA was purified as a single oligomeric species of 160–170 kDa, suggesting that it is a pentamer. All the bacterial strains, plasmids and phage used in this study are listed in Table 1. For standard culture of the organism, E. coli was grown aerobically in Erlenmeyer flasks filled to a maximum 10% of their volume with Luria–Bertani (LB) medium on a rotary shaker (250 r.p.m.) and by incubation at 37 °C. Anaerobic growths for the membrane fraction isolation were also performed at 37 °C either in Hungate tubes for small-scale CT99021 datasheet cultures or in sealed bottles filled with anaerobic medium and under a nitrogen gas atmosphere. Cultures were grown in LB supplemented with 0.4% w/v glucose. Cultures for the determination of β-galactosidase activity of lacZ fusions were grown in buffered TGYEP medium, pH 6.7, either without or with supplementation of 50 mM sodium formate (Begg et al., 1977). For overproduction of FocA variants, cells of BL21 (DE3) containing the appropriate plasmid were grown in TB medium, which included 0.12% w/v tryptone, 2.4% w/v yeast extract, 0.4% w/v glycerol, 0.4% w/v glucose, 170 mM KH2PO4, 72 mM K2HPO4, 2 mM MgSO4 and 0.37% w/v aspartic
acid. All media were supplemented with 0.1% v/v standard trace element solution (Hormann & Andreesen, 1994). The antibiotics kanamycin CX 5461 and ampicillin, when Baricitinib required, were added to the medium at the final concentrations of 50 and 100 μg mL−1, respectively. Genomic DNA from E. coli MC4100 was isolated using the Qiagen DNeasy Blood and Tissue
kit. The amplification of the focA gene was carried out using the primers focAIBA5f (5′-ATG GTA GGT CTC AGC GCC AAA GCT GAC AAC CCT TTT GAT CTT T-3′) and focAIBA5r (5′-ATG GTA GGT CTC ATA TCA ATG GTG GTC GTT TTC ACG CAG G-3′) or focAIBA3f (5′-ATG GTA GGT CTC AAA TGG TGA AAG CTG ACA ACC CTT TTG AT-3′) and focAIBA3r (5′-ATG GTA GGT CTC AGC GCT ATG GTG GTC GTT TTC ACG CAG G-3′), in each case introducing Eco31I restriction sites. The resulting fragments were cloned into a pASK-IBA5 /pASK-IBA3 vector (http://www.IBA-GO.com). The resulting plasmids pASK-IBA5focA/pASK-IBA3focA expressed focA derivatives that had an N-terminal or a C-terminal Strep-tag, respectively. A 252-bp DNA fragment including the fdhF promoter and regulatory region (Rossmann et al., 1991) was amplified from the chromosome of MC4100 using the primers fFDHEco (5′-GGGGAATTCAGTTGATGAAATCGCTGG-3′) and fFDHBam (5′-GGGGATCCAAATCACGCATACGCGCTC-3′), and after digestion with BamHI and EcoRI, was cloned into EcoRI–BamHI-digested pRS551 (Simons et al., 1987). Transfer of the insert to λRS45 and then to the chromosome of various strains was performed as described (Sawers & Böck, 1989). Anaerobic cultures were harvested at an OD600 nm of approximately 0.8.