Implementation and Testing of PURE mRNA Display

描述(申请人提供)：迫切需要开发识别蛋白质表面的新分子工具，用于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，作为传感器)到蛋白质组学(用于质谱学或基于芯片的诊断的亲和试剂)到新的治疗线索。在这里，我们建议实现和测试一种新的组合方法来构建自然和非自然的mRNA展示文库-纯mRNA展示(PURE=使用重组元素进行蛋白质合成)。在这种方法中，蛋白质合成设备将由纯化的成分组装而成，提供对浓度和成分的完全实验控制。因此，与经典的mRNA展示相比，纯mRNA展示具有几个潜在的优势，最重要的是为构建含有非天然氨基酸的设计者展示文库提供了一条简便的途径。我们的具体目标是：1.验证是否有可能利用纯蛋白质合成系统构建信使核糖核酸-肽融合。2.在体外定向进化实验中比较纯的和基于Retic的mRNA展示。

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.