Production of Fusion Protein SpA:: EcoRI in Batch Culture in a 60‐L Airlift Tower Loop Reactor

Abstract

Escherichia coli JM103 carrying the expression plasmid pMTC48, repressor plasmid pRK248, and protection plasmid pEcoR4 was grown in a 60-L working volume airlift tower loop reactor on M9 minimal medium. Production of fusion protein SpA::EcoRI was induced by a temperature shift from 30 to 38 (optimum), 40, or 42 degrees C. The following parameters were monitored: cell mass concentration (X), total cell counts (TCC), number of colony-forming units (CFU), concentrations of glucose, acetate, ethanol, pyruvate, lactate, succinate, amino acids, and ammonia, and soluble and total protein content, as well as product concentration (enzyme activity of the fusion protein), dissolved oxygen concentration, oxygen utilization rate (OUR), CO2 production rate (CPR), respiration quotient (RQ), and volumetric mass-transfer coefficients (kLa). Product formation by temperature shift was only observed if LB concentrate was added to the culture at the same time the aeration rate was increased to avoid oxygen-transfer limitation. No product accumulation was observed with glucose and ammonia supplementation. During gene expression, X and TCC increased, CFU decreased, acetate increased, and the primary metabolite (ethanol, pyruvate, lactate) concentrations as well as OUR and CPR passed a maximum while RQ changed only slightly. These facts indicate that, during gene expression, the metabolic activity of the cell passes a maximum, and after that it decreases. With increasing aeration rate, the volumetric productivity increased, but the specific productivity with respect to the cell concentration decreased.

Metabolic enhancement due to plasmid maintenance

“…Because the difference of growth pattern between ~VI109 without plasmid cultured in minimal and complex media was so dramatic experiments were carried out with }M109 carrying no or three plasmids to test for the requirement of trace elements, vitamins and aminoacids. The addition of trace elements (Brandes et al, 1993) or vitamins (thiamine and biotin) did not show growth enhancement. However, as we show in Figure 4, the addition of proline induced a significant enhancement of the growth.…”

Process Development in Biotechnology - A Re-Evaluation

“…Even during the growth phase, the fraction of plasmid bearing bacteria can reduce considerably in spite of the presence of antibiotics [5,6]. Therefore, the plasmid bearing bacterium grows slower than the plasmid free host, which gradually expels the plasmid bearing ones.…”

Prozessentwicklung in der Biotechnologie – Ein Rückblick

“…Die optimale Konzentration von IPTG steigt mit dem Reaktorvolumen an. Diese Induktionen kommen wegen der hohen Kosten von IPTG und der geringen Geschwindigkeit der Temperaturerhöhung in groûen Reaktoren nicht in Frage [5].…”