A Collaborative Proposal: Engineering Disulfide Formation Kinetics to Enhance Heterologous Secretion in Saccharomyces Cerevisiae

含有二硫键的异源蛋白的分泌没有充分发挥其潜力。有证据表明，内质网氧化还原状态的调节可能是一个瓶颈。为了进一步研究瓶颈假说，并最终提高分泌效率，我们提出了一种多方面的方法来探测和管理酵母的氧化还原状态。提出的工作的四个要素是：(1)扩增目前被认为参与氧化还原管理的单个基因的产物以评估其影响；(2)使用显示方法发现可能缺失的途径元件；(3)向生长培养基中添加氧化还原改变化合物以改变氧化还原状态；(4)开发基于速型蛋白I底物的诊断系统。

The secretion of heterologous proteins that contain disulfide bonds is not at its full potential. Evidence suggests that the regulation of the redox state of the endoplasmic reticulum may be a bottleneck. A multifaceted approach is proposed to probe and manage the redox state in yeast in order to investigate the bottleneck hypothesis further and to ultimately increase secretion efficiency. The four elements of the proposed work are: (1) amplify the products of the individual genes now thought to be involved redox management to assess the effect, (2) use display methods to uncover possibly missing pathway elements, (3) add redox altering compounds to the growth medium to alter redox state, and (4) develop a tachyplesin I substrate-based diagnostic system.