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。它通常涉及生成一个蛋白质变异体的文库，并通过选择或筛选来识别那些具有所需特性的变异体。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几个数量级，允许对蛋白质进行更大的序列空间采样。目前还没有有效的体外方法来选择具有所需突变的蛋白质，以增强其热稳定性。耐热蛋白质可以在高温下发挥作用，通常是坚固的，在各种条件下耐降解，因此在广泛的工业和医疗应用中具有很高的价值。本项目旨在开发一种体外选择蛋白质热稳定性的技术。我们计划通过(1)构建一个重组的体外蛋白质合成系统(热纯系统)来实现这一目标，该系统使用来自嗜热菌的纯化成分，该细菌在720℃的最适温度下生长，以及(2)将该系统应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该项目的第一阶段(第一阶段)，并建立了一个初步的热纯系统，该系统允许在高达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目标蛋白来实现热纯系统的商业化。我们还建议将热纯系统与体外选择技术相结合，如体外区划和核糖体展示，以定向进化来自中温来源的耐热蛋白质。我们计划为各种酶和蛋白质测试几种定向进化的选择方案。如果成功，该项目将带来以下独特和有价值的商业产品：(1)蛋白质热合成试剂盒；(2)提高热稳定性的工程蛋白质服务；(3)耐热核酸酶作为研究界的新试剂和用于治疗应用的耐热单链抗体。

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.