High-Throughput Platforms to Study Synthetic Receptors, Natural Molecules, and New Pathway Inhibitors

项目摘要 计算和实验蛋白质工程的进展开启了生物分子的新纪元 开发，提供新的功能结合蛋白，改进的酶和合理设计的合成 感受器。尽管取得了这些进展，但现有的技术缺乏开发和研究合成受体的能力。 和高通量的抑制分子，而不是依赖于理性或功能不可知的工程 所需活动的低吞吐量表征所遵循的方法。这种方法上的缺陷 结果是1)开发出不太理想的候选蛋白质，2)开发过程成本高昂。 我的长期目标是1)建立新的平台，将蛋白质功能作为一种选择压力 设计新的分子和2)利用这些新的平台来理解天然和合成的作用 正常和疾病病理中细胞信号反应中的蛋白质。我猜想 将蛋白质功能作为选择压力纳入蛋白质工程活动将导致 高效开发能够回答关键生物学问题的新型功能蛋白质。这个 本次建议期的目标是建立高通量的抑制剂筛选平台 工程和合成受体工程，以及开发联合计算和 实验蛋白质工程管道，以协助重要蛋白质的研究。继续在 所描述的领域具有巨大的潜力，通过1)极大地帮助基础和合成生物学的研究 扩大分子的可获得性，使对细胞中单个分子重要性的研究成为可能 信号反应，2)为理解合成受体功能提供新的工具包，以及3)提供 模块化平台，以帮助发现功能工程分子。我们将朝三个主要方向努力： 方向一：建立酵母菌表面展示的栓系抑制剂工程平台。 目前的定向进化方法通常缺乏蛋白质作为选择压力的功能，导致大多数 被动结合蛋白的研究进展。我们将结合小分子系绳的概念 筛选群落以驱动酵母展示蛋白的选择走向活性抑制。 方向2.建立计算和实验相结合的蛋白质工程管道。 目前的蛋白质工程方法通常依赖于广泛的突变或深入的 计算/理性设计以开发功能蛋白质。我们将结合无障碍计算 蛋白质突变文库设计的方法，并研究这些方法用于基础突变研究。 方向三：建立高通量合成受体研究平台。合成的 受体在基础和临床科学中都产生了可测量的影响，但还没有有效的平台来 研究它们在高通量方面的发展。我们将建立高通量筛查平台， 受体的增殖为设计新的合成受体提供了一种选择压力。

Project Summary Advances in computational and experimental protein engineering have ushered in a new era of biomolecule development, providing new functional binding proteins, improved enzymes, and rationally designed synthetic receptors. Despite such progress, established techniques lack the ability to develop and study synthetic receptors and inhibitory molecules in high throughput, instead relying on rational or function-agnostic engineering approaches followed by low-throughput characterization for the desired activity. This deficiency in approach results in 1) the development of suboptimal candidate proteins and 2) a costly development process. My long-term goals are to 1) establish new platforms that incorporate protein function as a selective pressure to engineer new molecules and 2) utilize these new platforms to understand the roles of natural and synthetic proteins in cell signaling responses in both normal and disease pathologies. I hypothesize that the incorporation of protein function as a selective pressure in protein engineering campaigns will result in the efficient development of new classes of functional proteins capable of answering key biological questions. The goals during this proposal period are to establish high-throughput screening platforms for inhibitor engineering and for synthetic receptor engineering, as well as develop combined computational and experimental protein engineering pipelines to aid the study of important proteins. Continued work in the described areas has immense potential to aid the study of basic and synthetic biology through 1) greatly expanding the availability of molecules to empower studies of the importance of individual molecules in cell signaling responses, 2) providing a new toolkit for understanding synthetic receptor function, and 3) providing modular platforms to aid discovery of functional engineered molecules. We will pursue three primary directions: Direction 1: Establish a tethered inhibitor engineering platform with yeast surface display. Current directed evolution approaches often lack protein function as a selective pressure, resulting in majority development of passive binding proteins. We will incorporate the concept of tethering from the small molecule screening community to drive yeast-displayed protein selection toward active inhibition. Direction 2. Establish combined computational and experimental protein engineering pipelines. Current protein engineering approaches typically rely on widespread mutagenesis or in-depth computational/rational design to develop functional proteins. We will incorporate accessible computational approaches for protein mutant library design and investigate these approaches for basic mutational studies. Direction 3. Establish a high-throughput platform for studying synthetic receptors. Synthetic receptors have made measurable impacts in both basic and clinical science, but no effective platform exists to study their development in high throughput. We will establish a high-throughput screening platform with proliferation from receptor functions as a selective pressure for engineering new synthetic receptors.