Implementation and Testing of PURE mRNA Display

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，作为传感器）到蛋白质组学（用于质谱或基于芯片的诊断的亲和试剂）到新的治疗线索。在这里，我们提出实施并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 =使用重组元素合成蛋白质）。在这种方法中，蛋白质合成机制将由纯化的成分组装，提供浓度和成分的完整实验控制。因此，与传统的mRNA展示相比，PURE mRNA展示有几个潜在的优势，最重要的是，它提供了一种简便的途径来构建含有非天然氨基酸的设计展示库。我们的具体目标是：1。测试是否有可能使用PURE蛋白合成系统构建mrna -肽融合体。2. 在体外定向进化实验中比较PURE和基于reticnet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.