Enhancement of Intracellular and Extracelluar Protein Production in Recombinant Yeast

保持质粒的高稳定性和高效的蛋白分泌对于提高重组酿酒酵母胞内和胞外蛋白的产量至关重要。本研究的主要目的是研究质粒的稳定性和克隆基因在复杂培养基中的表达。只要选择合适的质粒，无论使用何种营养培养基，都可以选择含有质粒的细胞。详细的实验计划已经描述，重点是培养基富集对克隆基因产物合成的影响。研究结果将表明，自动选择系统的潜力，以生产异种蛋白。提出的工作的另一个重点是蛋白质分泌。将操纵各种遗传和环境因素来确定它们对蛋白质分泌效率的影响。特别是，启动子强度和转录速率的影响，诱导蛋白质分泌和复杂介质中的分泌将被研究。对于这两个领域的研究，最初的工作将是实验性的。可以预见，某些研究途径将适用于动力学建模。

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.