In vitro Reconstitution of Protein Translation of Thermus Thermophilus for Direct

嗜热栖热菌蛋白质翻译的体外重建用于直接

• 批准号: 7609535
• 负责人: Shaorong Chong
• 金额: $ 23.31万
• 依托单位: NEW ENGLAND BIOLABS, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7609535
• 关键词: Amino Acyl-tRNA Synthetases; Antibodies; Bacteria; Cell-Free System; Cells; Code; DNA-Directed RNA Polymerase; Enzymes; Escherichia coli; Evolution; Genes; Goals; In Vitro; Libraries; Medical; Methods; Natural regeneration; Peptide Hydrolases; Phase; Property; Protein Biosynthesis; Proteins; Ribosomes; Screening procedure; System; Technology; Temperature; Thermus thermophilus; Translations; base; directed evolution; experience; nuclease; overexpression; protein reconstitution; public health relevance; reconstitution; restriction enzyme; thermostability; translation factor

DESCRIPTION (provided by applicant): Directed protein evolution is a powerful technology for generating proteins with desired properties, such as thermostability. There is currently no effective in vitro method to select proteins with enhanced thermostability. Thermostable proteins have a wide range of industrial and medical applications. The aim of this project is to construct a reconstituted in vitro protein synthesis system (thermoPURE system) using purified components from Thermus thermophilus, a bacterium that grows at an optimal temperature of 72oC. The thermoPURE system may allow protein synthesis to be conducted in vitro at elevated temperatures, and due to its low background activities, facilitate in vitro screening and selection or other applications that require elevated temperatures. In conjunction with powerful in vitro selection technologies such as in vitro compartmentalization and ribosome display, the thermoPURE may become the only system that allows in vitro directed evolution of thermostable proteins from their mesophilic origins. During the initial phase of the project, we will clone a complete set of genes for the translation factors, aminoacyl-tRNA synthetases and energy regeneration enzymes from Thermus thermophilus. All proteins will be overexpressed and purified from E. coli. The ribosomes and tRNAs will be purified from a growing culture of Thermus thermophilus. Using our experience and capacity of manufacturing the E. coli version of the reconstituted protein synthesis system (the PURE system), we will reconstitute the protein translation machinery of Thermus thermophilus and demonstrate in vitro protein synthesis at elevated temperatures. In the second phase, we will apply the thermoPURE system for in vitro evolution of mesophilic proteins such as restriction enzymes, RNA polymerases, and single-chain antibodies with an ultimate goal to generate valuable proteins with enhanced thermostability for industrial and medical usage. PUBLIC HEALTH RELEVANCE: Proteins that are stable at elevated temperatures have a wide range of industrial and medical applications. There is currently no effective in vitro method to select proteins with enhanced thermostability. The goal of this project is to provide such a method by constructing a reconstituted in vitro protein synthesis system from purified components of Thermus thermophilus, a bacterium that grows at an optimal temperature of 72oC.

描述（由申请人提供）：有向蛋白质进化是一种强大的技术，用于生成具有所需特性的蛋白质，例如热稳定性。当前没有有效的体外方法可以选择具有增强热稳定性的蛋白质。热稳定蛋白具有广泛的工业和医疗应用。该项目的目的是使用Thermus Thermophilus的纯化成分构建一个重构的体外蛋白质合成系统（热pure蛋白系统），该细菌在最佳温度为72oC的最佳温度下生长。热电系统可以使蛋白质合成在升高的温度下进行体外进行，并且由于其背景活动较低，促进了体外筛查和选择或其他需要升高温度的应用。结合强大的体外选择技术，例如体外隔室化和核糖体显示，热pure可能成为唯一允许体外定向热稳定蛋白从其嗜嗜熟源中促进热稳定蛋白的系统。在该项目的初始阶段，我们将为Thermus Thermophilus的翻译因子，氨基酰基-TRNA合成酶和能量再生酶的完整基因克隆。所有蛋白质将过表达并从大肠杆菌中纯化。核糖体和TRNA将从嗜热嗜热培养物中纯化。利用我们制造重构蛋白质合成系统（纯系统）的大肠杆菌版本的经验和能力，我们将重建热嗜热的蛋白质翻译机械，并在高温下展示体外蛋白质合成。在第二阶段，我们将应用热掌系统，以在诸如限制酶，RNA聚合酶和单链抗体之类的嗜熟蛋白的体外演化，其最终目标可以产生具有增强热稳定性的有价值的蛋白质，以增强工业和医疗用途。 公共卫生相关性：在升高温度下稳定的蛋白质具有广泛的工业和医疗应用。当前没有有效的体外方法可以选择具有增强热稳定性的蛋白质。该项目的目的是通过从Thermus Thermophilus的纯化成分中构建一个重构的体外蛋白质合成系统来提供这种方法，Thermus Thermophilus的纯化成分是一种在72oC最佳温度下生长的细菌。