Engineering Disulfide Formation Kinetics to Enhance Heterologous Secretion in Saccharomyces cerevisiae (Collaborative)

9531407 Wittrup该提案是关于更好地理解体内二硫键形成过程并因此最大化异源蛋白分泌的研究。 在这种情况下，它提出了操纵氧化折叠在内质网（ER）的酵母酿酒酵母。 三个方面将研究其对异源分泌的影响：（1）两个关键的ER氧化还原折叠酶（PDI，Eug1p）的水平的优化，（2）新的小分子氧化还原折叠催化剂的开发和测试，以及（3）天然ER小分子量氧化还原缓冲液，谷胱甘肽的浓度和氧化还原电位的改变。 这些操作对异源分泌的影响将与表达和分泌系统进行研究：牛胰胰蛋白酶抑制剂，一个小的58个氨基酸的蛋白质与三个二硫键，和RNA酶A，一个124个残基的酶，含有4个二硫键。 ***

9531407 Wittrup This proposal is on research to better understand the disulfide formation process in vivo and to consequently maximize heterologous protein secretion. In this context, it is proposed to manipulate oxidative folding in the endoplasmic reticulum (ER) of the yeast Saccharomyces cerevisiae. Three aspects will be studied with respect to their effects on heterologous secretion: (1) optimization of the levels of two key ER redox foldases (PDI, Eug1p), (2) development and testing of novel small molecule redox folding catalysts, and (3) alteration of the concentration and redox potential of the natural ER small molecular weight redox buffer, glutathione. The effects of these manipulations on heterologous secretion will be studied with expression and secretion systems for: bovine pancreatic trypsin inhibitor, a small 58 amino acid protein with three disulfide bonds, and RNase A, a 124- residue enzyme that contains 4 disulfide bonds. ***