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

项目总结 在药物研究和开发中，对多肽药物的兴趣越来越大 (研发)因为多肽被认为是高度选择性和有效的，同时 相对安全，耐受性好。多肽连接酶在化学-酶促多肽合成中的应用 具有优良的纯度和收率，因此成为一种有吸引力的方法来取代传统的化学 一种多肽类药物的合成方法。然而，发展的高效和多功能 多肽连接酶滞后，限制了多肽疗法的进步。从根本上说，这是 由于目前依赖于合理蛋白质的多肽连接酶工程方法效率低下 工程学与低通量酶特性相结合。我们将克服这一限制 通过开发用于高通量多肽连接酶工程的专用微流控系统。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相结合 以实现多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，并展示其提高能力 枯草杆菌酶(一种特性良好的多肽连接酶)的氨解与水解比。在具体目标1中， 我们将开发一种用于超高通量和定量分析的微流控系统 枯草杆菌酶。我们将开发微流控硬件、流程和分析方法来实现分析 以及筛选大量(超过106)的枯草杆菌酶变异体。在具体目标2中，我们将 建立并测试枯草杆菌酶筛选平台。我们将设计和合成枯草杆菌酶变异体 库，我们将使用模拟演示我们的微流控系统的吞吐量和灵敏度 枯草杆菌酶变异体文库。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，高灵敏度的多肽连接酶试验可以与液滴结合。 微流控组件使多肽连接酶的高通量工程成为可能。A拟议的第二阶段 该项目将涉及筛选枯草杆菌酶库，以提高氨解与水解率 枯草杆菌酶及其工程体系在多肽其他重要性质工程中的应用 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。

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.