FORMULATION AND OPTIMIZATION OF 2 CULTURE MEDIA FOR THE PRODUCTION OFTUMOR NECROSIS FACTOR-BETA IN ESCHERICHIA-COLI

Two culture media were designed and optimized by statistical technique s for the growth of Escherichia coli K12 HB101 (pCG402) which expresse d human tumour necrosis factor-beta (TNF-beta). Common compounds, such as MgSO4 and KH2PO4, were used to provide the bacteria with the neces sary elements for biomass synthesis. For compounds not required for bi omass synthesis, such as thiamine, or compounds used for both biomass synthesis and as an energy source, such as glucose, yield coefficients were used. In formulating the composition of the nutrients, carbon wa s chosen as the limiting substrate in both media. In the complex mediu m (CM), casein hydrolysate was selected to supply the two auxotrophic amino acids, proline and leucine. The optimal ratio of glucose to case in hydrolysate was determined to be 1:0.6 by using a centre composite design experiment. In the defined medium (DM), the concentrations of t he three carbon sources, glucose, proline and leucine were based on th eir respective yield coefficients (Y-biomass/glucose: 0.5, Y-biomass/p roline: 7, Y-biomass/leucine: 13.5). Shake flask experiments based on a fractional design were used to confirm that the two media were gluco se limiting. Bacterial growth was improved in CM whereas DM gave highe r TNF-beta expression. A 2-dm(3) fed-batch fermentation using CM was p erformed and a dry biomass concentration of 20 g dm(-3) was obtained w ith the expression of soluble TNF-beta being 20 mg g(-1) dry biomass. With this fed-batch system, a high biomass concentration and high expr ession of TNF-beta were achieved concomitantly.