A novel biotherapeutic expression platform in bacteria

细菌中的新型生物治疗表达平台

• 批准号: 6880357
• 负责人: MATTHEW P DELISA
• 金额: $ 10万
• 依托单位: VYBION, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6880357
• 关键词: Escherichia coli; acidity /alkalinity; arginine; bacterial proteins; bioreactors; biotechnology; biotherapeutic agent; drug design /synthesis /production; hydrogen transport; membrane transport proteins; molecular chaperones; protein engineering; protein folding; protein transport; recombinant proteins; secretory protein; temperature

DESCRIPTION (provided by applicant): The overall objective of the proposal is to develop a novel protein expression platform by capitalizing on the remarkable properties of the recently discovered twin-arginine translocation (Tat) pathway of bacteria. With over 300 therapeutic proteins currently in various stages of clinical trials, the road to a healthier future will require new methods for producing safer and less expensive recombinant proteins. For this purpose, the recently discovered twin-arginine translocation (Tat) pathway will be utilized as a novel recombinant protein expression platform in Escherichia coli. Towards this objective, Phase I encompasses the following specific aims: 1) to define the secretory capacity of the bacterial Tat transporter; 2) to optimize Tat secretion efficiency via chaperone co-expression strategies; and 3) to scale-up the Tat-based protein production process. This research is expected to not only result in the development a novel platform for bacterial protein expression but will also facilitate a deeper understanding of a poorly understood biological mechanism. Phase II will entail a much broader effort to study and improve Tat transport efficiency including, for instance, directed evolution strategies to engineer "hyperactive" Tat transporters, cell engineering methods to optimize the host for Tat expression and leader peptide optimization studies. In addition, we expect to fully develop scale-up methods for large-scale Tat expression by using optimized carbon-source feed strategies, as well as temperature, dissolved oxygen and pH optimization.

描述（由申请人提供）：该提案的总体目标是利用最近发现的细菌双精氨酸易位（Tat）途径的显着特性来开发一种新型蛋白质表达平台。目前有超过 300 种治疗性蛋白质处于临床试验的不同阶段，通往更健康的未来之路将需要新的方法来生产更安全、更便宜的重组蛋白质。为此，最近发现的双精氨酸易位（Tat）途径将被用作大肠杆菌中的新型重组蛋白表达平台。为了实现这一目标，第一阶段包括以下具体目标：1）确定细菌 Tat 转运蛋白的分泌能力； 2) 通过伴侣共表达策略优化Tat分泌效率； 3) 扩大基于 Tat 的蛋白质生产工艺。这项研究不仅有望开发出细菌蛋白表达的新平台，还将有助于更深入地了解人们知之甚少的生物机制。第二阶段将需要更广泛的努力来研究和提高 Tat 转运效率，包括例如设计“过度活跃”Tat 转运蛋白的定向进化策略、优化 Tat 表达宿主的细胞工程方法和前导肽优化研究。此外，我们期望通过使用优化的碳源进料策略以及温度、溶解氧和 pH 值优化，全面开发用于大规模 Tat 表达的放大方法。