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

9604563 Raines这项建议正在进行研究，以更好地了解体内二硫化物的形成过程，从而最大限度地增加异源蛋白质的分泌。在此背景下，有人提议操纵酿酒酵母内质网(ER)中的氧化折叠。将从三个方面研究它们对异源分泌的影响：(1)优化两种关键的内质网氧化还原折叠酶(PDI，Eug1p)的水平；(2)新型小分子氧化还原折叠催化剂的开发和测试；(3)天然内质网小分子氧化还原缓冲液谷胱甘肽浓度和氧化还原电位的变化。这些操作对异源分泌的影响将通过表达和分泌系统来研究：牛胰蛋白酶抑制物，一个含有三个二硫键的58个氨基酸的小蛋白，以及RNaseA，一个包含四个二硫键的124个残基的酶。***

9604563 Raines 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. ***