Fig. 6

Western blot analysis of CYP21A2 and redox proteins in the whole-cell system for determination of the stoichiometric ratio of the three proteins encoded by the tricistronic construct p21b_ArEt. Western blot analysis was performed with polyclonal antibodies against a etp1^fd (11.3 kDa), b arh1 (51 kDa), c bovine CYP21A2 (54.6 kDa). In each blot, lane 1 represents the E. coli cell extract expressing arh1, etp1^fd and CYP21A2 after 28 h. a lane 2–8 represents purified etp1^fd in increasing amounts (10, 20, 30, 40, 50, 75 and 100 ng), b, c The lanes 2–7 show purified arh1 and CYP21A2 in increasing amounts (arh1: 25, 50, 75, 125, 187.5 and 250 ng, CYP21A2: 24, 48, 95, 143, 191 and 239 ng). The with “M” marked lanes all represent a prestained protein marker. The relative lane intensities, which correlate with the respective protein masses, were determined and compared to the intensity of the whole-cell system sample. Mass values were converted into the amount of substances and extrapolated to the expression yield per liter culture. Note that etp1^fd is known to give a single band in the range of the double mass expected to see on the SDS-PAGE