Fig. 4

Characterizing in the cytoplasmic membrane produced YidC-GFP and GltP-GFP. BL21(DE3)pETyidC-gfp and BL21(DE3)pETgltP-gfp cells cultured in the absence and presence of IPTG as described in the legend of Fig. 3 were harvested and membranes were isolated. a The quality of produced YidC-GFP fraction that was inserted into the cytoplasmic membrane was judged by the FSEC profiles of DDM-solubilised membranes. The FSEC trace of YidC-GFP purified from cells cultured in the presence of IPTG is in grey (90.3 µg of total protein was loaded containing 0.29 μg of YidC-GFP) and the FSEC trace of YidC-GFP purified from cells cultured in the absence of IPTG is represented in black (25.4 µg of total protein was loaded containing 0.43 μg of YidC-GFP) (relative fluorescence unit, RFU). Traces were normalized according to the dilution factor used to obtain equivalent fluorescence intensities prior to solubilisation of the membranes (see “Methods”). b GltP-GFP was purified from the membranes and incorporated in liposomes, and glutamate uptake was determined. As a control, liposomes without reconstituted protein were used. Activity measurements of GltP-GFP purified from cells cultured in the absence of IPTG are represented in black and activity measurements in plain liposomes are represented in red. Note that the amount of GltP-GFP produced in BL21(DE3) cells cultured in the presence of IPTG was insufficient to determine activity