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

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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. ***