Fig. 5

pReX enables the use of a helper plasmid for the efficient production of a cytochrome c (OCC) in the periplasm. BL21(DE3)/pReXompAocc and BL21(DE3)/pETompAocc cells also harboring pEC86 were cultured in LB medium at 30 °C. The expression of the gene encoding secretory OCC was induced with 400 μM IPTG for 24 h. l-rhamnose was present as indicated. a The effect of the production of secretory OCC following varying gene expression levels on biomass formation was monitored by measuring the A₆₀₀. b OCC production in equal amounts of cells isolated from the cultures described in A was monitored using immuno-blotting with an antibody against the Strep-tag. Accumulation levels of the T7 lysozyme, when appropriate, and IbpB in cells producing OCC were monitored by immuno-blotting using antibodies against T7 lysozyme and IbpB, respectively. c The OCC was isolated using Strep-tag-based purification from BL21(DE3)/pReX/pEC86 cells cultured at the optimal production condition and BL21(DE3)/pET/pEC86 cells from 3 L cultures as described in the “Methods” section. Left panel: the OCC isolation from BL21(DE3)/pReX/pEC86 cells cultured at the optimal l-rhamnose (the tube on the left) resulted in material with a distinct red color under ambient light, which is indicative for mature cytochrome c. 0.24 mg of protein was isolated from 1 l of culture. The colour of the material isolated from BL21(DE3)/pET/pEC86 cells (the tube on the left) was much lighter. 0.04 mg of protein was isolated from 1 l of culture. Right panel: optical spectra of the material shown in the left panel recorded corroborated that much more mature OCC was produced using the BL21(DE3)/pReX/pEC86 setup compared to the BL21(DE3)/pReX/pEC86 setup