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Recombinant protein production in E.coli

Recombinant protein production in E.coli. Molecular and process strategies. Plasmid maintenance and copy number. Plasmid stability : Segregational stability Structural stability Host cell mutation. Improving plasmid stability. Programmed cell death upon loss of plasmid

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Recombinant protein production in E.coli

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  1. Recombinant protein production in E.coli Molecular and process strategies

  2. Plasmid maintenance and copy number Plasmid stability : Segregational stability Structural stability Host cell mutation

  3. Improving plasmid stability • Programmed cell death upon loss of plasmid • Integrate gene of interest in chromosome – gene copy number ? • Combination of selection pressure and growth rate control by limiting the carbon source in the medium

  4. Expression vector design consideration

  5. High Cell Density cultivation • Development of growth media • Acetate formation • Oxygen concentration • Mixing patterns in large scale fermenters • Dissolved carbondioxide

  6. Above the following concentrations E.coli is inhibited. Glucose – 50 g/l Ammonia – 3 g/l Phosphorus – 10 g/l Magnesium – 8.7 g/l Zinc -0.038 g/l Iron 1.15 g/l Hence fed batch is the most preferable method

  7. General methods applied to reduce acetate formation in E. coli

  8. General methods applied to reduce acetate formation in E. coli

  9. Oxygen concentration • Pure oxygen can be sparged. But expensive • Anaerobic conditions lead to acetate formation • Mixing • Cells close to the injection port are exposed to high concentrated nutrients.

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