Replication of the Human Secretory Pathway in Yeast

酵母中人类分泌途径的复制

• 批准号: 6883699
• 负责人: STEFAN WILDT
• 金额: $ 73.17万
• 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 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6883699
• 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): With its ability to secrete high levels of recombinant proteins, the yeast Pichia pastoris is extensively used for protein manufacturing. However, as actual yields vary widely for individual proteins, GlycoFi is focusing on the improvement of protein yields in P. pastoris as part of the company's long term mission to develop yeast-based protein production technology for manufacturing therapeutic proteins with human glycosylation structures. In Phase I of this project a strategy was developed and tested using the model protein alpha 1-antitrypsin for overcoming bottlenecks in yeast secretion that result in less than optimal yields. A combinatorial library/high throughput screening methodology was designed to optimize translocation of the nascent peptide into the endoplasmic reticulum (ER) and protein folding within the ER. In Phase II, yield optimization studies will include IgG as well as alpha 1-antitrypsin with specific experiments targeting expression and secretion of the IgG H and L chains. The toolkit created in Phase I will be expanded through the implementation of several new high throughput approaches toward yield improvement including mutagenesis and cDNA library screening and deletion of protease genes as well as traditional fermentation and purification process development. Proposed Commercial Application: GlycoFi's Humanized Yeast(tm) enables the study of glycosylation structure-function relationships and creates the ability not only to pinpoint the specific glycoforms that optimize the bioactivity, pharmacokinetics and overall efficacy of a therapeutic protein, but also to manufacture them at low cost with a high degree of uniformity. 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）：由于其分泌高水平重组蛋白的能力，酵母巴斯德毕赤酵母广泛用于蛋白质生产。然而，由于单个蛋白质的实际产量差异很大，GlycoFi专注于提高巴斯德毕赤酵母的蛋白质产量，作为公司长期使命的一部分，开发基于酵母的蛋白质生产技术，用于生产具有人类糖基化结构的治疗性蛋白质。在该项目的第一阶段，开发了一种策略，并使用模型蛋白α 1-抗胰蛋白酶进行了测试，以克服导致产量低于最佳产量的酵母分泌瓶颈。设计组合文库/高通量筛选方法以优化新生肽到内质网（ER）中的易位和ER内的蛋白质折叠。 在II期，产量优化研究将包括IgG以及α 1-抗胰蛋白酶，并进行针对IgG H和L链表达和分泌的特定实验。在第一阶段创建的工具包将通过实施几种新的高通量方法来扩大产量提高，包括诱变和cDNA文库筛选和蛋白酶基因的缺失以及传统的发酵和纯化工艺开发。建议的商业应用：GlycoFi的Humanized Yeast（TM）能够研究糖基化结构-功能关系，不仅能够精确定位优化治疗性蛋白质生物活性、药代动力学和整体功效的特定糖型，还能够以低成本和高度均匀性制造它们。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，更经济的方式进入临床。