In Vitro Selection of Proteins via mRNA-Protein Fusions

分子设计中最具挑战性的问题之一是开发分子， 可以结合蛋白质表面并阻断蛋白质-蛋白质相互作用。调节的能力 以定向方式的蛋白质-蛋白质相互作用开启了探测和 通过设计控制生物系统这项建议的长远目标 是利用体外选择实验来靶向蛋白质表面， 蛋白质识别，结构，功能，特异性和催化方面。体外 遗传学方法目前代表了一种强有力的蛋白质操作解决方案， 设计问题。此前，PI已经构思、开发和实施了mRNA- 肽和蛋白质融合物（下文称为“mRNA展示"），以使用体外 选择实验mRNA展示相对于其他基因表达技术提供了重要的优势。 体外和体内蛋白质设计策略，如检查非常大的能力， 文库（>1013个单独的序列），在不存在活细胞的情况下， 对结合和严格性的实验控制。在这一建议中，我们将继续 使用G蛋白连接的信号传导作为配体设计的目标。我们的目标是探索 生物物理化学的蛋白质识别，因为它涉及到这一重要的信号 通路我们的具体目标是： 1)为了增强我们的G蛋白和GPCR定向配体的性质。 2)开发靶向2-肾上腺素能受体的肽和蛋白质配体， (2AR)G蛋白偶联受体（GPCR）。 3)探索我们的GPCR定向配体的功能和结构。

One of the most challenging problems in molecular design is developing molecules that can bind protein surfaces and block protein-protein interactions. The ability to modulate protein-protein interactions in a directed fashion opens the possibility of probing and controlling biological systems through design. The long- term objective of this proposal is to use in vitro selection experiments to target protein surfaces, exploring fundamental aspects of protein recognition, structure, function, specificity, and catalysis. In vitro genetic approaches currently represent a powerful operational solution to the protein design problem. Previously, the PI has conceived, developed, and implemented mRNA- peptide and protein fusions (hereafter "mRNA display) to design proteins using in vitro selection experiments. mRNA display provides important advantages relative to other in vitro and in vivo protein design strategies, such as the ability to examine very large libraries (>1013 individual sequences) in the absence of a living cell, with tight experimental control over binding and stringency. In this proposal, we will continue using G-protein linked signaling as a target for ligand design. Our goal is to explore the biophysical chemistry of protein recognition as it pertains to this important signaling pathway. Our specific aims are: 1) To enhance the properties of our G protein and GPCR-directed ligands. 2) To develop peptide and protein ligands targeting the 2-adrenergic (2AR) G protein coupled receptor (GPCR). 3) To explore the function and structure of our GPCR-directed ligands.