A platform for engineering peptide ligase for building next generation peptide therapeutics.

用于构建下一代肽疗法的肽连接酶工程平台。

• 批准号: 9908228
• 负责人: Adam R. Abate
• 金额: $ 22.5万
• 依托单位: SCRIBE BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-07 至 2021-08-06
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9908228
• 关键词: Abate; Academia; Attention; Biochemistry; Biological Assay; Case Study; Collaborations; Coupled; Cysteine; Development; Engineering; Enzymes; FDA approved; Fluorescence Resonance Energy Transfer; Goals; Hydrolysis; Institution; Kinetics; Lead; Libraries; Ligase; Ligation; Mediating; Medicine; Methods; Microfluidics; Modernization; Molecular Biology; Mutagenesis; Mutation; N-terminal; Nature; Optics; Peptide Synthesis; Peptides; Performance; Pharmacologic Substance; Phase; Process; Production; Property; Protein Engineering; Proteins; Reaction; Renaissance; Research; Resources; Services; Sorting - Cell Movement; Specificity; Structure; System; Testing; Time; Variant; base; chemical synthesis; design; experience; experimental study; high throughput screening; improved; instrument; interest; manufacturing process; member; next generation; novel; peptide drug; racemization; research and development; screening; sortase; subtiligase

PROJECT SUMMARY There is an increased interest in peptide medicines in pharmaceutical research and development (R&D) because peptides are recognized as highly selective and efficacious, and at the same time relatively safe and well tolerated. Chemo-enzymatic peptide synthesis (CEPS) using peptide ligases features excellent purity and yield, thus becoming an attractive method to replace traditional chemical synthesis method for synthesizing peptide drugs. However, the development of efficient and versatile peptide ligases lags behind, limiting the advancement of peptide therapeutics. Fundamentally, this is due to the inefficiency of current peptide ligase engineering method that relies on rational protein engineering coupled with low throughput enzymatic characterization. We will overcome this limitation by developing a specialized microfluidic system for high throughput peptide ligase engineering. We will merge modern biochemistry and molecular biology methods with advanced droplet microfluidics to enable high throughput screening of peptide ligase variants. In this project, we will build an enzyme screening platform and demonstrate its capacity on increasing aminolysis to hydrolysis ratio of subtiligase (a well characterized peptide ligase). In Specific Aim 1, we will develop a microfluidic system for ultrahigh-throughput and quantitative analysis of subtiligases. We will develop the microfluidic hardware, processes, and assays to enable the analysis and screening of a large number (over 106) of variants of subtiligase. In Specific Aim 2, we will establish and test subtiligase screening platform. We will design and synthesize subtiligase variant library and we will demonstrate the throughput and sensitivity of our microfluidic system using a mock subtiligase variant library. We will also establish kinetic assays for charactering subtiligase. Achieving these aims will prove that peptide ligase assay with high sensitivity can be incorporated with droplet microfluidic components to enable high throughput engineering of peptide ligase. A proposed phase II project would involve screening of subtiligase libraries to increase aminolysis to hydrolysis ratio of subtiligase and applying our engineering system to engineer other important properties of peptide ligases such as substrate selectivity and racemization activity as well as early production of hardware and disposables.

项目摘要 对肽药物在药物研究和开发中的兴趣增加了 （研发）由于肽被认为是高度选择性和有效的，同时又 相对安全且耐受性良好。使用肽连接酶的化学酶肽合成（CEP） 具有出色的纯度和产量，因此成为替代传统化学化学物质的一种有吸引力的方法 合成肽药物的合成方法。但是，有效和通用的发展 肽连接酶滞后，限制了肽疗法的进步。从根本上讲，这就是 由于当前肽连接酶工程方法的效率低下，该方法依赖于理性蛋白 工程和低通量酶促表征结合。我们将克服这个限制 通过开发用于高通量肽连接酶工程的专门微流体系统。我们 将合并现代生物化学和分子生物学方法与先进的液滴微流体学 为了使肽连接酶变体的高吞吐量筛选。 在这个项目中，我们将建立一个酶筛选平台，并展示其增加的能力 枯草酶的氨基溶解与水解酶的水解比（一种表征良好的肽连接酶）。在特定的目标1中， 我们将开发一个微流体系统，用于超高通知和定量分析 枯草酶。我们将开发微流体硬件，过程和测定，以实现分析 并筛选大量的枯草酶变体（超过106）。在特定的目标2中，我们将 建立和测试枯草酶筛选平台。我们将设计和合成枯草酶变体 库，我们将使用模拟来证明微流体系统的吞吐量和灵敏度 枯草酶变体库。我们还将建立用于表征枯草酶的动力学测定。实现 这些目标将证明具有高灵敏度的肽连接酶测定可以与液滴合并 微流体成分可以使肽连接酶的高吞吐量工程。提出的II期 项目将涉及筛查枯草酶库以增加氨基溶解与水解比的水解比 枯竭酶并将我们的工程系统应用于肽的其他重要特性 诸如底物选择性和种族化活动以及硬件的早期生产等连接酶 和一次性。