Produção de proteinas recombinantes utilizando Escherichia coli em cultivos em alta densidade celular

AUTOR(ES)
DATA DE PUBLICAÇÃO

2004

RESUMO

This thesis studied submerged high cell-density cultures (HCDC) of recombinant Escherichia coli identifying important kinetic parameters on growth and production phases. Several strategies to obtain HCDC were studied aiming to develop a robust fermentation process that allowed: High cell dry weight per liter; to determine the nutritional conditions to express the fusion protein in levels up to 20% of total protein, and to determine the best fermentation technique to reach dry biomass concentration higher that 100,0 g/L. The strain used in this work was E. colí N-4830-1 transformed with the plasmid pHis. The fermentations were ran out in 4.0 L and 12.0 L bench-top bioreactors (NBS e Inceltech). Total protein concentration, recombinant protein concentration, cell concentration, glucose concentration and acetate concentration were analyzed. The kinetics studies demonstrated that biomass and fusion protein were increased, however the best results were performed on a continuous fermentation with microfiltration, total cell recycle and exponencial feeding. The cell productivity was PR = 6,437 glUh, the final cell dry weight was 173,8 g/L and the percentage of fusion protein was 17,8 % of total protein. The biomass and fusion protein increase obtained were, respectively, 341 % and 328 %, comparing with traditional fed-batch fermentation. This technique showed appropriated to remove inhibitors of the broth allowing higher cell concentration and, consequently, higher productivity. In this type of culture strategy, the final cell concentration was 3.5 fold than one obtained in traditional fed-batch

ASSUNTO(S)

biotecnologia - microbiologia microbiologia industrial dna recombinante membranas (tecnologia) fermentação

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