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 在药物研究和开发中，对肽类药物的兴趣越来越大 （R&D）因为肽被认为是高度选择性和有效的，同时 相对安全且耐受性良好。使用肽连接酶的化学-酶促肽合成（CEPS） 具有纯度高、收率高的特点，是一种很有吸引力的替代传统化学合成方法。 一种合成肽类药物的合成方法。然而，开发高效、通用的 肽连接酶滞后，限制了肽治疗剂的发展。从根本上说，这是 由于目前依赖于合理蛋白质的肽连接酶工程方法效率低下， 工程化结合低通量酶表征。我们将克服这一限制 通过开发用于高通量肽连接酶工程的专用微流体系统。我们 将现代生物化学和分子生物学方法与先进的液滴微流体技术相结合， 以实现肽连接酶变体的高通量筛选。 在这个项目中，我们将建立一个酶筛选平台，并展示其在增加 枯草杆菌连接酶（一种充分表征的肽连接酶）的氨解与水解比率。具体目标1、 我们将开发一种用于超高通量和定量分析的微流控系统， 枯草杆菌气体。我们将开发微流控硬件、流程和分析，以实现分析 以及筛选大量（超过106种）枯草杆菌连接酶的变体。在第二阶段，我们将 建立并测试枯草杆菌酶筛选平台。我们将设计并合成枯草杆菌酶变体 库，我们将证明我们的微流控系统的吞吐量和灵敏度使用模拟 枯草杆菌酶变体文库。我们还将建立枯草杆菌酶的动力学分析方法。实现 这些目的将证明具有高灵敏度的肽连接酶测定可以与液滴结合， 微流体组件，以实现肽连接酶的高通量工程化。拟议的第二阶段 该项目将涉及筛选枯草杆菌蛋白酶文库，以增加蛋白酶的氨解与水解比率。 枯草杆菌蛋白酶和应用我们的工程系统工程肽的其他重要性质 连接酶如底物选择性和外消旋活性以及硬件的早期生产 和一次性用品。