Rossi, Gregory P; (1995) The production and purification, by an affinity method, of a secreted glycosylated fusion protein from Saccharomyces cerevisiae. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The system employed in this work consisted of a bacterial β-lactamase gene fused to the secretion and promotion regions of the homologous Saccharomyces cerevisiae SUC2 gene which included the coding for the first 30 amino acid residues of yeast invertase. Escherichia coli β-lactamase was used as it is a prokaryotic product and therefore contains no eukaryotic sorting signals and no sites for fortuitous glycosylation, it is also adapted to crossing membranes as it is secreted to the periplasm in E. coli. Investigations into maximizing product formation using the transformed strain DBY746.1 showed a differential in the product obtained between complex and minimal media in the fermenter culture although expression levels in both systems were identical. This phenomenon was shown to be due to interfacial effects and not proteolytic degradation resultant from to nutrient limitation in minimal media. The degradation was observed at both shake flask and fermenter scales and was prevented by the addition of surfactants. A variety of methods designed to increase the yield of the fusion protein revealed a relationship between growth rate and both expression and secretion of the heterologous product. It was shown that mid logarithmic phase cells do not only have greater expression levels but also a greater secretion ability dependant on the rate of growth. The growth and induction systems were scaled up from shake flask to 1001 pilot plant scale. These systems gave enhanced secreted product yield over shake flask induction systems. Having produced enough protein for characterization work investigations into a purification system were made. After initial routine purification steps proved unsuccessful, an immuno-affinity chromatography stage was developed to facilitate separation of the protein of interest. The pure product was then subjected to initial glycan characterization confirming the linkage and chain type and number of chains.

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