In vitro Reconstitution of Protein Translation of Thermus Thermophilus for Direct

描述（由申请人提供）：定向蛋白质进化是蛋白质工程的强大技术。它通常涉及生成蛋白质变体库，并通过选择或筛选来鉴定具有所需特性的蛋白质变体。就文库复杂性而言，体外选择方法比基于细胞的选择方法优越上级几个数量级，允许对蛋白质中大得多的序列空间进行采样。目前没有有效的体外方法来选择具有增强其热稳定性的所需突变的蛋白质。热稳定蛋白质可以在高温下发挥作用，通常在各种条件下具有鲁棒性和抗降解性，因此对于广泛的工业和医学应用是有价值的。本项目旨在开发一种体外筛选蛋白质热稳定性的技术。我们计划通过（1）使用来自嗜热栖热菌（Thermus thermophilus）（一种在720 ℃的最佳温度下生长的细菌）的纯化组分构建重构的体外蛋白质合成系统（thermo PURE系统），以及（2）应用这种系统定向进化具有增强的热稳定性的蛋白质来实现这一目标。 我们已经成功地完成了该项目的第一阶段（第一阶段），并建立了一个初始的热纯系统，允许在高达600 ℃的温度下体外合成活性全长蛋白质。对于第二阶段，我们建议通过优化系统和测试更多的靶蛋白来商业化thermo PURE系统。我们还建议使用热纯系统结合体外选择技术，如在体外区室化和核糖体展示，从他们的嗜温起源的热稳定蛋白质的定向进化。我们计划测试多种酶和蛋白质的定向进化的几种选择方案。如果成功，该项目将产生以下独特和有价值的商业产品：（1）蛋白质热合成试剂盒;（2）工程蛋白质热稳定性增强服务;（3）热稳定核酸酶作为研究界的新试剂和热稳定单链抗体用于治疗应用。 公共卫生相关性：蛋白质可以被工程化以在高温下发挥功能，并且在各种条件下表现出稳健性和抗降解性。该项目旨在开发一种技术，以选择工程热稳定蛋白质用于工业和医疗用途。

DESCRIPTION (provided by applicant): Directed protein evolution is a powerful technology for protein engineering. It generally involves generating a library of protein variants and identifying those with desired properties by selection or screening. In terms of library complexity, in vitro selection methods are superior to cell-based selection methods by several orders of magnitude, allowing a much larger sequence space in proteins to be sampled. There is currently no effective in vitro method to select proteins with desired mutations that enhance their thermostability. Thermostable proteins can function at high temperatures, are generally robust and resistant to degradation under a variety of conditions, and therefore are valuable for a wide range of industrial and medical applications. This project intends to develop a technology for in vitro selection of protein thermostability. We plan to achieve this goal by (1) constructing a reconstituted in vitro protein synthesis system (thermo PURE system) using purified components from Thermus thermophilus, a bacterium that grows at an optimal temperature of 720C, and (2) applying such system for directed evolution of proteins with enhanced thermostability. We have successfully completed the first phase of the project (Phase I) and established an initial thermo PURE system that allowed in vitro synthesis of active full- length proteins at temperatures up to 600C. For the Phase II, we propose to commercialize the thermo PURE system by optimizing the system and testing more target proteins. We also propose to use the thermo PURE system in conjunction with in vitro selection technologies, such as in vitro compartmentalization and ribosome display, for directed evolution of thermostable proteins from their mesophilic origins. We plan to test several selection schemes of directed evolution for a variety of enzymes and proteins. If successful, this project would lead to the following unique and valuable commercial products: (1) a protein thermo synthesis kit; (2) a service for engineering proteins with enhanced thermostability; (3) thermostable nucleic acid enzymes as new reagents for research communities and thermostable single-chain antibodies for therapeutic applications. PUBLIC HEALTH RELEVANCE: Proteins can be engineered to function at high temperatures and exhibit robustness and resistance to degradation under a variety of conditions. This project intends to develop a technology to select engineered thermostable proteins for industrial and medical use.