Recombinant protein production (RPP) is a cornerstone of bioprocessing. This study presents novel analytical techniques and production protocols for RPP in E. coli, particularly regarding industrial applications. Flow cytometry (FCM) was used to monitor cell physiology and RPP during production of a fluorescent model protein, CheY::GFP. Further applications of FCM for monitoring RPP were developed: The amyloidophilic dye Congo red was used to identify inclusion bodies produced under high-stress conditions. FCM analysis of transformants on agar plates identified 3 populations of varying fluorescence intensity and the progressive transfer of cells from the high fluorescence population to one of intermediate fluorescence and low culturability. Congo red staining showed this was due to amyloid-inclusion body formation. RPP conditions that minimise physiological stress by reducing temperature and inducer concentration can increase product yields, solubility and biomass yields. The original fermentation protocol used for stress-minimised RPP proved unsuitable for industrial use. Application of stress-minimisation to an industrially-derived protocol using early or late-phase induction and glucose or glycerol as carbon source generated high biomass, total CheY::GFP and soluble CheY::GFP yields. These protocols improved biomass generation, product formation and reproducibility over the original stress-minimised and unmodified industrially-derived protocols and therefore stress-minimisation is of potential industrial use.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:642384 |
| Date | January 2015 |
| Creators | Wyre, Christopher John |
| Publisher | University of Birmingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://etheses.bham.ac.uk//id/eprint/5743/ |
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