The maintenance of high plasmid stability and efficient protein secretion are important for the enhancement of intracellular and extracellular protein production in recombinant Saccharomyces cerevisiae. A major objective of the research outlined in this proposal is to study plasmid stability and cloned gene expression in complex media. With the proper choice of a plasmid, selection for plasmid-containing cells occurs regardless of the nutrient medium used. A detailed experimental plan has been described, focusing on the effects of medium enrichment on cloned gene product synthesis. The results of the research will indicate the potential of autoselection systems for the production of heterologous proteins. Another primary emphasis of the proposed work is protein secretion. Various genetic and environmental factors will be manipulated to determine their influence on the efficiency of protein secretion. In particular, the influence of promoter strength and transcription rate, the induction of protein secretion, and secretion in complex media will be investigated. For both areas of research, the initial work will be experimental. It is anticipated that certain avenues of research will be amenable to kinetic modeling.
