FIG. 5.
Programming and monitoring optimized cultivation protocols. (A) Monitoring of growth and temperature in a culture of BL21(DE3)(pDIA17) expressing the Rv0289 gene of M. tuberculosis. The culture was grown in automated mode with preprogrammed temperature shifts and addition of inducer. ▪, temperatures; ▴, OD600. The arrow indicates the time of IPTG addition. (B) Analysis of soluble recombinant protein produced without and with a temperature downshift to 14°C. Cells harvested at the end of the culture were subjected to analytical purification as described in Materials and Methods. Lanes 1 to 5, fractions from a culture maintained at 30°C for 3 h after induction; lanes 6 to 10, fractions from the culture shown in panel A, which was shifted to 14°C, induced, and then incubated overnight at the same temperature; lanes 1 and 6, total soluble extract; lanes 2 and 7, insoluble fraction; lanes 3 to 5 and 8 to 10, fractions purified by metal affinity chromatography.