EFFICIENT SCREEN FOR INTERACTING NEURONAL GENE PRODUCTS

信号通路的复杂性使人们认识到蛋白质-蛋白质相互作用的多样性和重要性，然而鉴定新的相互作用基因产物的方法既耗时又有限。这项建议的目标是开发一种方法，用于高通量筛选结合伙伴的多个目标蛋白，并快速鉴定它们结合的结构域。我们正在开发的方法学是基于在噬菌体衣壳表面表达的cDNA文库。批量分离直接与靶蛋白结合的含噬菌体的多肽序列。然后，模拟目标蛋白中特定序列的重叠合成肽被用作噬菌体结合的靶标，以识别特定的结合域。这一新的范式是由两个最新的进展实现的：1.在噬菌体T7中开发了一个表达系统，它将主要衣壳蛋白的编码序列与约150个碱基对的cDNA插入物融合在一起；2.开发了一个半自动系统，在该系统中，多肽被共价连接到96个“针”支撑物上，大大简化了目标残基的高通量鉴定。该系统可以很容易地扩展到384个“针”或更大，并且可能是完全自动化的。该建议有两个步骤或目标：第一，完善克隆和表达最佳长度的cDNA的系统，以及测试它们与特定蛋白质和合成肽靶标相互作用的能力。其次，通过识别与髓鞘结构蛋白相互作用的新分子，并验证它们的表达模式，来测试该系统的多功能性。我们选择髓鞘特异性蛋白作为实验靶点，因为这些蛋白的突变会导致严重的髓鞘脱失表型。因此，这些研究将第一次允许在人类疾病状态下单一氨基酸替换和效应器结合丧失之间建立强大的相关性。

The complexity of signaling pathways has led to an appreciation of the diversity and importance of protein-protein interactions, yet the methodologies for identification of novel interacting gene products are time consuming and limited in scope. The goal of this proposal is to develop a methodology for high throughput screening of multiple target proteins for binding partners, and rapid identification of the domain to which they bind. The methodology we are developing is based on libraries of cDNAs which are expressed at the capsid surface of bacteriophage. Phage bearing peptide sequences that bind directly to target proteins are isolated in batch. Overlapping synthetic peptides mimicking specific sequences in the target protein are then used as targets for phage binding to identify specific binding domains. This novel paradigm is made possible by two recent advances: 1. The development of an expression system in phage T7 which fuses the coding sequence for the major capsid protein with cDNA inserts of approximately 150 base pairs, and 2. the development of a semi-automated system in which peptides are synthesized covalently attached to a 96 "pin" support, dramatically simplifying high throughput identification of target residues. This system is readily expandable to 384 "pin"or greater and is potentially fully automatable. The proposal has 2 steps or aims: First, perfection of the system for cloning and expressing optimal length cDNAs, as well as testing their ability to interact with defined protein and synthetic peptide targets. Second, testing the versatility of the system by identifying novel molecules that interact with myelin structural proteins for which partners have yet to be identified and verification of their expression pattern. We have chosen myelin specific proteins as experimental targets, as mutations in these proteins cause severe dysmyelinating phenotypes. Thus for the first time, these studies will permit powerful correlations to be made between single amino acid substitutions and the loss of effector binding in human disease states.