A two step procedure for successive periods of protein synthesis and folding. (a) Affinity chromatography purified proteins were separated by SDS-PAGE and stained with SimplyBlue (Invitrogen). Soluble recombinant proteins Btke, Kringle and Chip (Sol) recovered after chaperone-induced folding in BL21(DE3) control cells and cells co-expressing the eight different chaperone combinations described in Fig. 1. Cells were subjected to the one-step (O) or two-step (T) protocols. The total amounts of recombinant protein (Tot) expressed in control and chaperone co-overproducing cells were evaluated after purification of the protein under denaturating conditions. (b, c) Optimization of the (re)folding conditions in step two for Btke using chaperone combination 4. (b) After overnight culturing at 20°C the cells were pelleted, resuspended in fresh medium and cultured for 1 h, 2 h, 3 h, and 4 h at 20°C, or 1 h and 2 h at 37°C in the presence of 200 μg/ml chloramphenicol. For each time point the one-step (O) and two-step (T) procedures were compared. (c) Purified Btke from uninduced cells (control) or cells induced with IPTG according to the one-step procedure (induced) were compared with cells subjected to the two-step procedure with varying conditions: IPTG-induced overnight cultures were pelleted and further grown in fresh media without addition (growth for 1, 2, 4 h) or with addition of chloramphenicol or IPTG (growth for 2 h).