The majority of proteins differentially expressed in E. coli were downregulated at 20°C, possibly due to stress induced by reducing the temperature after culturing. At 37°C, most of the proteins were upregulated over time. The samples taken at 20°C and time point t4 showed similarities to those taken at the early time point and 37°C (Figure 3). A possible explanation for this is that the cultures had adapted to the low temperature and started to produce similar proteins to the 37°C cultures.
Different downstream processing conditions were analyzed by fractionating the collected samples and using either a Capto™ Q anion exchange column or a HisTrap™ IMAC HP column. Different fractions were collected from each column and analyzed by 2-D DIGE. Chromatograms from the two purification methods are shown in Figure 4A. Two examples of protein spot maps of samples cultured at 37°C and fractionated using either ion exchange or affinity chromatography are shown in Figure 4B.
Because all gels in the experiment used the same internal standard, it was possible to link the spot maps of the fractionated samples back to the spot maps of the start material (cultured at 37°C or 20°C). The spot maps showed that several host-cell proteins were still present after IMAC purification. These were likely to be histidine- or tryptophan-containing proteins, which can also bind to the resin. Many variants/isoforms of the histidine-tagged target protein could also be separated and identified. Using information obtained from 2-D DIGE it is possible to reduce certain impurities in the eluate by selecting optimal upstream and downstream conditions.