Development of scale-down techniques for investigation of recombinant Escherichia coli fermentations: Acid metabolites in shake flasks and stirred bioreactors
| Autor: | George Hugh A, Alan L. Powell, Randolph Greasham, Mary Ellen Dahlgren |
|---|---|
| Rok vydání: | 1993 |
| Předmět: |
Recombinant Fusion Proteins
Metabolite Carboxylic Acids Exotoxins Buffers Biology medicine.disease_cause complex mixtures chemistry.chemical_compound Laboratory flask Escherichia coli medicine Bioreactor chemistry.chemical_classification Chromatography Ethanol Immunotoxins Hydrogen-Ion Concentration Transforming Growth Factor alpha equipment and supplies Carbon Recombinant Proteins Culture Media Oxygen Glucose chemistry Biochemistry Fermentation Aeration Biotechnology Organic acid |
| Zdroj: | Biotechnology Progress. 9:580-586 |
| ISSN: | 1520-6033 8756-7938 |
| DOI: | 10.1021/bp00024a003 |
| Popis: | We have developed shake-flask screening conditions that are predictive of specific expression of the chimeric toxin, TGFα-PE40, by recombinant Escherichia coli JM109 in stirred bioreactors. When a nutrient-rich stirred bioreactor medium was used in shake flasks, neither the extent of growth nor the specific level of recombinant protein expression duplicated the performance in stirred bioreactor fermentations. Incomplete oxidation of glucose and concomitant accumulation of organic acid metabolites, as well as oxygen limitation and lack of pH control, were examined as contributors to the poorer performance in the flask. The medium buffering capacity, initial glucose level, and flask aeration were evaluated to establish the limits of “scale-down” conditions for expression both in a complex nutrient medium (M101) similar to that used in stirred bioreactors and in a defined (FM) medium. Acid metabolites and ethanol were measured as indicators of carbon flow from glucose as well as indirect indicators of oxygen limitation. For the complex M101 medium, optimal shake-flask performance in 250-mL, nonbaffled flasks at 37°C occurred with 0.3X medium strength, supplementation with 0.3 M HEPES buffer (pH 7.5), and 10 mL of medium per flask. Cultures grown under these conditions produced a maximum density of 3.6 g of dry cell weight/L (as estimated by absorbance measurements at 600 nm) and maintained a pH near neutrality. Additionally, metabolite markers of anaerobic or microaerobic conditions, such as ethanol, lactate, and pyruvate, were not detected, and specific expression of TGFα-PE40 was comparable to stirred bioreactors induced for expression at various biomass levels. When culture parameters were controlled within these limits, similar results were also observed in the defined FM medium. |
| Databáze: | OpenAIRE |
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