Automatic inducer addition and harvesting of recombinant Escherichia coli cultures based on indirect on-line estimation of biomass concentration and specific growth rate

This article describes a novel bioreactor configuration for production opti mization of recombinant proteins in Escherichia coli, Inducer addition and harvesting are controlled on-line based on indirect estimation of biomass c oncentration and specific growth rate from addition of NaOH to maintain con stant pH. When either a predetermined biomass concentration is reached or t he cultures have obtained, a constant specific growth rate inducer is intro duced automatically, The induction period is ended by automatic harvesting of the cultures either at a predetermined biomass concentration or when sub strate (in this study glucose) is depleted, detected as an increase of pH, or dissolved oxygen tension. During harvesting, metabolic activities are qu enched within 3 min by cooling of the cell suspension. The system has been used to optimize expression of glutathione S-transferase (GST) fusion prote in of the ligand binding domain of mouse peroxisome proliferator-activated receptor, GST-PPAR alpha LBD. Total yield of GST-PPAR alpha LBD was indepen dent of the time of inducer addition as long as the length of induction per iod corresponded to at least 0.25 cell divisions while the yield of soluble GST-PPAR alpha LBD, the only active form, increased with the length of ind uction period. Highest yields were obtained when the inducer was added at l ow cell concentration as soon as constant specific growth rate was detected , resulting in induction periods corresponding to 3.4 +/- 0.4 cell division s. The specific growth rate remained almost constant for one cell division after inducer addition, whereafter it decreased. No decrease of specific gr owth rate was observed when inducer was added in the lag-phase, and no solu ble protein was produced. These results suggest that solely soluble GST-PPA R alpha LBD acts as a growth inhibitor and that GST-PPAR alpha LBD is expre ssed predominantly as inclusion bodies immediately after inducer addition w hereas the proportion expressed as soluble protein is increased after 1 h o f induction, Compared to the procedures, which are generally used for prote in expression in the laboratory, this system is less labor intensive, it au tomatically provides recording of biomass concentration and specific growth rate, and it allows direct comparisons between expression of different pro teins and performance of different constructs since the induction period is linked to growth. (C) 2001 John Wiley & Sons, Inc.