Replication of the Human Secretory Pathway in Yeast

酵母中人类分泌途径的复制

• 批准号: 6791073
• 负责人: STEFAN WILDT
• 金额: $ 10万
• 依托单位: GLYCOFI, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-20 至 2004-10-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6791073
• 关键词: alpha 1 antitrypsin; biotechnology; biotherapeutic agent; cell line; drug design /synthesis /production; endoplasmic reticulum; fungal proteins; high throughput technology; method development; molecular biology information system; molecular chaperones; molecular cloning; peptide library; polymerase chain reaction; protein engineering; protein folding; protein signal sequence; recombinant proteins; secretory protein; synthetic protein; transport proteins; yeasts

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.

描述（由申请人提供）：尽管毕赤酵母以其分泌高水平重组蛋白的能力而闻名，但每种蛋白质的实际产量差异很大。因此，GlycoFi的长期目标是专注于提高P. pastoris的蛋白质产量，作为公司使命的一部分，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。这个第一阶段项目的目标是开发和测试一种策略，以克服分泌过程中导致产量低于最佳产量的瓶颈，特别关注改善新生肽进入内质网（ER）的易位和内质网内的蛋白质折叠。该项目的目标是：1)建立一个真菌信号序列文库；2)建立一个人类伴侣蛋白文库；3)开发一种筛选方法来测试这些信号序列和伴侣蛋白是否会增加模型蛋白的分泌。综上所述，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。该工具包将在二期高通量筛选中用于识别优化产量的信号序列和伴侣蛋白的组合。拟议的商业应用：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一种高容量的替代哺乳动物细胞培养的方法，使改善生命的药物以更快、更经济的方式进入临床。