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Recombinant protein production using E. coli containing temperature-sensitive constructs

Osborne, Neil A; (1990) Recombinant protein production using E. coli containing temperature-sensitive constructs. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The principles of large scale process design for the use of E. coli strains containing temperature sensitive constructs were developed. Two strains were characterised in a 14L fermenter, one containing a constant copy number plasmid (pCQV2), and the other an amplifiable copy number plasmid (pMG169). Batch fermentations were performed under kinetic limitation to determine key parameters such as optimum conditions for growth and production, oxygen requirements, and the effects of different lengths of time for the implementation of the temperature shift. These results suggested that on the large scale oxygen limitation was likely to occur. Therefore fermentations were performed with a limiting oxygen transfer rate typical of a 100m3 reactor (50-60 mM.L-1h-1. Batch productivity under oxygen limitation fell by between 84% and 87%. The negative effects of oxygen limitation were removed by fermenter operation under glucose limitation, which yielded maximum batch productivity of the three strategies. At all times the strain containing plasmid pMG169 yielded higher batch productivities than the one containing pCQV2. The heat of fermentation was measured (using a continuous calorimetric method) as a means of partially implementing the temperature shift. The results differed from those obtained from widely used empirical correlations (which should not be used with recombinant strains). Design calculations were performed that allow batch and annual titres to be predicted under different fermenter configurations, scales, and modes of operation.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Recombinant protein production using E. coli containing temperature-sensitive constructs
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Batch fermenters
URI: https://discovery.ucl.ac.uk/id/eprint/10120380
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