초록
The kinetics of recombinant human granulocyte-macrophage colony-stimulating factor (hGM-CSF) expression was studied under the strong T7 promoter in continuous culture of Escherichia coli using complex medium to design an optimum feeding strategy for high cell density cultivation. Continuous culture studies were done at different dilution rates and the growth and product formation profiles were monitored post-induction. Recombinant protein expression was in the form of inclusion bodies with a maximum specific product formation rate (q<SUB>p</SUB>) of 63.5 mg g<SUP>-1</SUP> DCW h<SUP>-1</SUP> at a dilution rate (D) of 0.3 h<SUP>-1</SUP>. The maximum volumetric product concentration achieved at this dilution rate was 474 mg l<SUP>-1</SUP>, which translated a ~1.4 and ~1.75 folds increase than the values obtained at dilution rates of 0.2 h<SUP>-1</SUP> and 0.4 h<SUP>-1</SUP> respectively. The specific product yield (Y<SUB>P/x</SUB>) peaked at 138 mg g<SUP>-1</SUP> DCW, demonstrating a ~1.6 folds increase in the values obtained at other dilution rates. A drop in q<SUB>p</SUB> was observed within 5-6 h of induction at all the dilution rates, possibly due to protein toxicity and metabolic stress associated with protein expression. The data from the continuous culture studies allowed us to design an optimal feeding strategy and induction time in fed-batch cultures which resulted in a maximum product concentration of 3.95 g l<SUP>-1</SUP> with a specific hGM-CSF yield (Y<SUB>P/x</SUB>) of 107 mg g<SUP>-1</SUP> DCW.