Fig. 3

Optimizing the production of the membrane protein GltP using pReX. BL21(DE3) cells harbouring pReXgltPgfp and Lemo21(DE3) cells harboring pETgltPgfp were cultured and target gene expression was induced as described in Fig. 2. a Left panel: to assess the effect of different l-rhamnose concentrations on GltP–GFP production levels, we monitored fluorescence (relative fluorescence unit, RFU) per milliliter of culture. Right panel. The effect of GltP-GFP production in the absence and presence of increasing concentrations of l-rhamnose on biomass formation was monitored by measuring the A₆₀₀. The production of GltP-GFP under Lemo21(DE3) and pReX-based optimal conditions corresponds to 9.6 and 9.7 mg of protein produced per liter of culture, respectively [25]. b Top panel: the integrity of the GltP–GFP fusion produced at different l-rhamnose concentrations was monitored using in-gel fluorescence. Bottom panel. Accumulation levels of IbpB in cells producing GltP–GFP at different l-rhamnose concentrations were monitored by immuno-blotting using an antibody against IbpB. c GltP-GFP was purified from the membranes of BL21(DE3)/pReX and Lemo21(DE3)/pET cells cultured at the optimal l-rhamnose concentration. Approximately 2.5 mg of GltP-GFP was isolated from 1 l of Lemo21(DE3) and pReX-based cultures. Equal amounts of isolated GltP-GFP were subsequently incorporated in liposomes, and glutamate uptake was determined. Plain liposomes were used as a control