Replication of the Human Secretory Pathway in Yeast

描述（由申请人提供）：尽管已知巴斯德毕赤酵母能够分泌高水平的重组蛋白，但单个蛋白的实际产量差异很大。因此，GlycoFi的长期目标是提高巴斯德毕赤酵母中的蛋白质产量，这是公司使命的一部分，即开发基于酵母和真菌的平台，用于生产具有人类糖基化结构的治疗性蛋白质。这个I期项目的目标是开发和测试一种克服分泌过程中导致产量低于最佳产量的瓶颈的策略，特别关注改善新生肽到内质网（ER）中的易位和ER内的蛋白质折叠。该项目的目标是1）组装真菌信号序列库，2）创建人类伴侣蛋白库，3）开发筛选方法来测试这些信号序列和伴侣蛋白以增加模型蛋白的分泌。总的来说，第一阶段的目标将导致创建一个工具包，用于在表达重组蛋白的酵母菌株中产生更像人类的分泌途径。该工具包将用于第二阶段的高通量筛选，以鉴定优化产量的信号序列和分子伴侣的组合。建议的商业应用：由于全球缺乏制造能力，阻碍了新疗法的开发管道，GlycoFi的技术将为生物制药行业提供哺乳动物细胞培养的高容量替代品，使改善生活的药物以更快，更经济的方式到达临床。

DESCRIPTION (provided by applicant): Although Pichia pastoris is known for its ability to secrete high levels of recombinant proteins, actual yields vary widely for individual proteins. Consequently, GlycoFi's longterm objectives are focused on the improvement of protein yields in P. pastoris as part of the company's mission to develop yeast and fungi-based platforms for the production of therapeutic proteins with human glycosylation structures. The goal of this Phase I project is to develop and test a strategy for overcoming bottlenecks in the secretory process causing less than optimal yields, focusing specifically on improving translocation of the nascent peptide into the endoplasmic reticulum (ER) and protein folding within the ER. The project aims are to 1) assemble a library of fungal signal sequences, 2) create a library of human chaperone proteins, and 3) develop a screening method to test these signal sequences and chaperone proteins for increased secretion of a model protein. Taken together, Phase I aims will result in the creation of a toolkit for producing a more human-like secretory pathway in yeast strains expressing a recombinant protein. The toolkit will be utilized in Phase II in a high throughput screen to identify combinations of signal sequences and chaperones that optimize yield. Proposed Commercial Application: With a worldwide lack of manufacturing capacity blocking the development pipeline for new therapeutics, GlycoFi's technology will provide the biopharmaceutical industry with a high capacity alternative to mammalian cell culture that will allow life-improving drugs to reach the clinic in a faster, more cost-effective manner.