Kirk, Niall Andrew; (1994) Process effectiveness of yeast expression vectors. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes work on aspects of specific regulated Saccharomyces cerevisiae promoters, namely, the growth phase and medium dependent regulation of the yeast heat-shock promoter element or HSE (Chapter 3); the promoter of the polyubiquitin gene (Chapter 4) and the promoter of the mating factor alpha, MFαl gene (Chapter 5). Chapter 3 describes the use of temperature upshift as a convenient way of inducing heterologous gene expression using a plasmid in which the regulatory elements from a yeast heat-shock promoter were present in a modified CYC1 promoter lacZ fusion in place of the CYC1 promoter regulatory region. Protein induction levels of up to 50 fold were seen on temperature upshift of a logarithmically growing yeast culture from 23°C to 39°C. Heat inducibility of the HSE was maximal at 39°C and lost at stationary phase of growth. The potential problem of protein degradation was tackled by using protease deficient strains which increased β-galactosidase accumulation 2-fold. Studies with the yeast heat shock promoter UBI4 (Chapter 4), chosen because of its potential use as a growth phase dependent promoter, showed that this promoter is primarily controlled (not as previously reported by intracellular cAMP levels) by carbon catabolite repression and this control is exerted probably through the HAP 2/3/4 regulatory system and as such is the first gene for a non-mitochondrial component shown to be controlled by this system. The yeast MFαl promoter was studied (Chapter 5). Results indicate that this promoter is regulated by the growth rate of the host cell, its activity rising as the cellular growth rate falls. Therefore maximum expression levels from this promoter can be achieved by growing cells at a low growth rate under respiratory conditions, a situation that will maximize both cell biomass and protein expression levels.

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