Enhancing Cell Density
The expression experiments for OmpG using HCDF were performed on nondeuterated, unlabeled media for optimization. The batch medium contained sufficient substrates (8 g/L glucose, 2 g/L NH4Cl) for growing the E. coli to OD600nm of about 8. Before induction, a biomass accumulating phase was introduced. First the effects of 0, 2, 4, and 6 h additional unlabeled feeds were studied. Six hours of additional cell growth from unlabeled substrates resulted in an OD600nm of about 13 (Figure 1).
The effects of enhancing cell density on the expression level were analyzed by SDS-PAGE. The specific protein production rate was kept constant over the time tested, and the generation of higher biomass leads to more recombinant protein yields.
The four deuterated HCDF set-ups for production of DCN-OmpG were programmed with a biomass accumulating feed that lasted 6 hours (Figure 2). After a prolonged lag phase the cells grew at an easy rate, finally attaining ODs comparable to the nondeuterated experiments. The automatic switch from batch to fed-batch was initiated by the rising dissolved oxygen (DO) once the substrates had depleted.
The signal triggered the start of DO-based feeding (deuterated medium with unlabeled N- and C-sources) for further biomass enrichment. The DO allowed adjustment to the requirements of the cells. After six hours the feed automatically switched to labeled feed. The feeding rate was kept constant for sufficient protein production. After one hour the IPTG as inducer for protein production was automatically added. The duration of expression was another six hours, after which the feeding was stopped by the process control. The reactors were cooled for later cell harvest.
The specific protein production rate was even better than that of the shaking culture, producing an 11-fold higher yield of protein from the same volume of culture.
Two different NMR-spectra are shown in Figure 3. Spectrum A reflects 15N labeling as 1H-15N correlations. The original deuterated nitrogens were protonated due to the purification procedure with water-containing buffers. Spectrum B shows missing 1H-13C-proton correlations, as deuterated carbon atoms keep the deuteron bound, even in water. The spots to be seen in spectrum B originated from the natural abundant 1H-13C-proton correlation of the nondeuterated detergent used for refolding.
These results show that the HCDF labeling protocol is a useful tool for 2H13C15N -labeled protein production under the requirements of NMR spectroscopy.