Investigation of recombinant protein production by Escherichia coli : expression of Green fluorescent protein and a co-factor dependent flavinated enzyme

摘要

This thesis summarises work done on the Escherichia coli strain MG1655 expressing audGreen Fluorescent Protein (GFP) and the flavo-protein N-methyl-L-tryptophan oxidaseud(MTOX) product and examining the effect foreign protein production has on cell growthudparameters. It also uses molecular modelling tools to generate data relating to FAD fluxudand MTOX production, comparable to that seen in E.coli fermentations.udThe MG1655 strain was chosen as it was the focus of the first K-12 complete sequencingudproject and closely related to the strain W3110, a second K strain that had been used touddevelop a number of deletion mutants which were central to the study.udIt presents data from shake flask and stirred tank reactor fermentations on minimal,udcarbon limited and complex media. Samples from these growth experiments were thenudanalysed concentrating on biomass concentration, protein assays (both chemical andudfluorimetric), high performance liquid chromatography and calculation of yieldudparameters. From this a baseline of growth was established with which to compareudchanges in growth after a shift in protein product from GFP to MTOX. An assay was alsouddeveloped to measure the amount of active and inactive MTOX enzyme produced andudthis data compared with the level of FAD available to the cell at specific time pointsudthroughout growth.udFinally modelling work is presented and in silico values compared with those generatedudin vitro. A discussion of the entire study concludes the work.