IN VITRO SELECTION OF PROTEINS VIA MRNA-PROTEIN FUSIONS

本提案中概述的实验旨在使用体外选择实验来探索蛋白质结构，识别和催化。 由于目前不可能设计出具有所需功能特性的肽或蛋白质序列，因此我们选择了体外遗传策略。 我们通过使用RNA-蛋白质融合物，肽或蛋白质分子共价连接到编码它们的mRNA上来实现这一点。 RNA-蛋白质融合允许蛋白质的重复选择和扩增，因为编码序列和多肽序列在单个分子中结合。该技术允许在完全不存在活细胞的情况下产生含有超过1013个不同序列的文库。 因此，融合提供了一种功能性的蛋白质设计方法，并提供了经典的遗传筛选不能应用的选择。 我们的具体目标是：1）提高我们对使用RNA-蛋白质融合进行蛋白质选择的理解和实施。 我们以前已经实现了RNA-蛋白质融合体作为体外肽和蛋白质选择的载体。 我们建议i）检查在核糖体上融合形成的机制ii）改进融合分子的合成和选择，和iii）在我们的组合文库的构建中探索蛋白质设计假设。 2)开发和探索调节信号转导途径的肽和蛋白质。 我们将使用体外选择实验来分离肽和蛋白质，模拟已知的G蛋白功能调节剂。 我们将检查我们选择的蛋白质与其靶G蛋白的亲和力，特异性和结构，比较我们分离的分子与天然调节剂的大小和多样性。 3)开发和探索新型催化剂的体外分离方法。我们将研究分离新的肽和蛋白质催化剂的策略。 分离序列的多样性，机制和结构将被探索，着眼于酶结构的基本问题。从这些实验中得到的信息具有巨大的潜力，可以教我们关于蛋白质结构、识别和催化的知识。 此外，我们的工作应该使其他实验室将其作为蛋白质发现和解剖的通用工具。 所使用的技术和分离的特定分子应极大地促进用于治疗人类疾病的疗法的开发。

The experiments outlined in this proposal are designed to use in vitro selection experiments to explore protein structure, recognition, and catalysis. Because it is not currently possible to design a peptide or protein sequence that is imbued with desired functional properties, we have chosen to pursue an in vitro genetic strategy. We do this by using RNA-protein fusions, peptide or protein molecules covalently attached to the mRNA which encodes them. RNA-protein fusions allow for repeated rounds of selection and amplification of proteins because the coding and polypeptide sequences are united in a single molecule. This technique allows libraries containing more than 1013 different sequences to be generated in the total absence of a living cell. Thus, fusions provide a functional approach to protein design and afford selection where classical genetic screening cannot be applied. Our specific aims are: 1) To improve our understanding and implementation of protein selection using RNA-protein fusions. We have previously implemented RNA- protein fusions as a vehicle for in vitro peptide and protein selection. We propose to i) to examine the mechanism of fusion formation on the ribosome ii) to improve the synthesis and selection of fusion molecules, and iii), to explore protein design hypotheses in the construction of our combinatorial libraries. 2) To develop and explore peptides and proteins that modulate signal transduction pathways. We will use in vitro selection experiments to isolate peptides and proteins that mimic known regulators of G protein function. We will examine the affinity, specificity, and structure of our selected proteins with their target G protein, comparing the size and diversity of the molecules we isolate with the natural regulators. 3) To develop and explore methods to isolate novel catalyts in vitro. We will examine strategies to isolate novel peptide and protein catalysts. The diversity, mechanism, and structure of the sequences isolated will be explored with an eye toward fundamental questions of enzyme architecture. The information that will result from these experiments has tremendous potential to teach us about protein structure, recognition, and catalysis. In addition, our work should enable other laboratories to apply it as a general tool for protein discovery and dissection. The techniques used and specific molecules isolated should greatly facilitate the development of therapeutics for the treatment of human disease.