In vitro Reconstitution of Protein Translation of Thermus Thermophilus for Direct

描述（申请人提供）：定向蛋白质进化是一项强大的蛋白质工程技术。它通常包括生成蛋白质变体库，并通过选择或筛选确定具有所需特性的蛋白质变体库。在文库复杂性方面，体外选择方法优于基于细胞的选择方法几个数量级，允许在蛋白质中取样更大的序列空间。目前还没有有效的体外方法来选择具有所需突变以增强其热稳定性的蛋白质。热稳定蛋白可以在高温下发挥作用，在各种条件下通常是坚固的和耐降解的，因此对广泛的工业和医疗应用是有价值的。本项目旨在开发一种热稳定性蛋白的体外筛选技术。为了实现这一目标，我们计划(1)利用从嗜热热菌（Thermus thermophilus，一种生长在720C的最佳温度下的细菌）中纯化的成分构建一个重组的体外蛋白质合成系统（thermo PURE系统），(2)将该系统用于具有增强热稳定性的蛋白质的定向进化。我们已经成功地完成了项目的第一阶段（一期），并建立了一个初始的thermo PURE系统，该系统可以在高达600℃的温度下体外合成活性全长蛋白。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实现热纯系统的商业化。我们还建议将thermo PURE系统与体外选择技术（如体外区隔化和核糖体展示）结合使用，用于从中温性起源中定向进化热稳定蛋白。我们计划测试几种针对多种酶和蛋白质的定向进化选择方案。如果成功，该项目将产生以下独特而有价值的商业产品：(1)蛋白质热合成试剂盒；(2)具有增强热稳定性的工程蛋白服务；(3)热稳定型核酸酶作为研究界的新试剂，热稳定型单链抗体用于治疗。

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.