Implementation and Testing of PURE mRNA Display

PURE mRNA 显示的实施和测试

• 批准号: 7025315
• 负责人: RICHARD W ROBERTS
• 金额: $ 24.45万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7025315
• 关键词: bioimaging /biomedical imaging; cell membrane; combinatorial chemistry; fusion gene; high throughput technology; image enhancement; messenger RNA; molecular /cellular imaging; molecular site; peptide analog; peptide library; protein binding; protein biosynthesis; protein localization; protein protein interaction; protein purification; proteomics; technology /technique development

DESCRIPTION (provided by applicant): There is a pressing need to develop new molecular tools that recognize protein surfaces for uses ranging from imaging probes to systems biology (blocking or modulating protein-protein interactions, serving as sensors) to proteomics (affinity reagents for mass spectrometry or chip-based diagnostics) to novel therapeutic leads. Here, we propose to implement and test a new combinatorial approach for constructing both natural and unnatural mRNA display libraries-PURE mRNA display (PURE = Protein synthesis Using Recombinant Elements). In this approach, the protein synthesis machinery will be assembled from purified components, providing complete experimental control of both concentrations and constituents. PURE mRNA display thus has several potential advantages relative to the classical mRNA display, most importantly in providing a facile route to construct designer display libraries bearing unnatural amino acids. Our specific aims are: 1. To test if it is possible to construct mRNA-peptide fusions using the PURE protein synthesis system. 2. To Compare and contrast PURE vs. retic-based mRNA display for in vitro directed evolution experiments.

描述（由申请人提供）：迫切需要开发新的分子工具来识别蛋白质表面，其用途从成像探针到系统生物学（阻断或调节蛋白质-蛋白质相互作用，用作传感器）到蛋白质组学（用于质谱或基于芯片的诊断的亲和试剂）到新型治疗先导物。在这里，我们建议实施和测试一种新的组合方法来构建天然和非天然 mRNA 展示库 - PURE mRNA 展示（PURE = 使用重组元件的蛋白质合成）。在这种方法中，蛋白质合成机器将由纯化的成分组装而成，提供浓度和成分的完整实验控制。因此，相对于经典的 mRNA 展示，PURE mRNA 展示具有几个潜在的优势，最重要的是提供了一种简便的途径来构建带有非天然氨基酸的设计展示文库。我们的具体目标是： 1. 测试是否可以使用 PURE 蛋白质合成系统构建 mRNA-肽融合体。 2. 比较和对比 PURE 与基于网状结构的 mRNA 显示，用于体外定向进化实验。