In Vitro Selection of Proteins via mRNA-Protein Fusions

描述（由申请人提供）：分子设计中最具挑战性的问题之一是开发能够结合蛋白质表面并阻断蛋白质-蛋白质相互作用的分子。以定向方式调节蛋白质-蛋白质相互作用的能力开启了通过设计探测和控制生物系统的可能性。本提案的长期目标是利用体外选择实验来靶向蛋白质表面，探索蛋白质识别，结构，功能，特异性和催化的基本方面。目前，体外遗传方法是解决蛋白质设计问题的有效方法。此前，PI已经构思，开发和实施mRNA肽和蛋白质融合（以下简称“mRNA展示”），通过体外选择实验设计蛋白质。相对于其他体外和体内蛋白质设计策略，mRNA展示提供了重要的优势，例如在没有活细胞的情况下检查非常大的文库（bbb1013个单个序列）的能力，以及对结合和严格性的严格实验控制。在本提案中，我们将使用G蛋白连接信号（特别是G蛋白a亚基和G蛋白偶联受体）作为配体设计的靶标。我们的目标是探索蛋白质识别的生物物理化学，因为它与这一重要的信号通路有关。我们的具体目标是：1)开发类和状态特异性G蛋白定向配体。2)测试G蛋白导向配体的功能。3)探索靶向玛土撒拉G蛋白偶联受体（GPCR）肽配体的结构和功能。4)开发针对趋化因子gpcr的新型抗体拟配体。

DESCRIPTION (provided by applicant): One (1) 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 use G-protein linked signaling (specifically G protein a subunits and G protein-coupled receptors) as targets 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 develop class- and state-specific G protein-directed ligands. 2) To test the function of our G protein-directed ligands. 3) To explore the structure and function of peptide ligands targeting the Methuselah G protein-coupled receptor (GPCR). 4) To develop novel antibody mimetic ligands targeting chemokine GPCRs.