Hudman, P.A.; (2011) Investigation of recombinant protein production by Escherichia coli : expression of Green fluorescent protein and a co-factor dependent flavinated enzyme. Doctoral thesis , UCL (University College London).

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Abstract

This thesis summarises work done on the Escherichia coli strain MG1655 expressing a Green Fluorescent Protein (GFP) and the flavo-protein N-methyl-L-tryptophan oxidase (MTOX) product and examining the effect foreign protein production has on cell growth parameters. It also uses molecular modelling tools to generate data relating to FAD flux and MTOX production, comparable to that seen in E.coli fermentations. The MG1655 strain was chosen as it was the focus of the first K-12 complete sequencing project and closely related to the strain W3110, a second K strain that had been used to develop a number of deletion mutants which were central to the study. It presents data from shake flask and stirred tank reactor fermentations on minimal, carbon limited and complex media. Samples from these growth experiments were then analysed concentrating on biomass concentration, protein assays (both chemical and fluorimetric), high performance liquid chromatography and calculation of yield parameters. From this a baseline of growth was established with which to compare changes in growth after a shift in protein product from GFP to MTOX. An assay was also developed to measure the amount of active and inactive MTOX enzyme produced and this data compared with the level of FAD available to the cell at specific time points throughout growth. Finally modelling work is presented and in silico values compared with those generated in vitro. A discussion of the entire study concludes the work.

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