EFFICIENT SCREEN FOR INTERACTING NEURONAL GENE PRODUCTS

信号通路的复杂性导致了对蛋白质-蛋白质相互作用的多样性和重要性的认识，然而鉴定新的相互作用基因产物的方法既耗时又有限。本提案的目标是开发一种高通量筛选多种靶蛋白结合伙伴的方法，并快速识别它们结合的结构域。我们正在开发的方法是基于在噬菌体衣壳表面表达的cdna文库。直接与靶蛋白结合的噬菌体携带肽序列分批分离。重叠的合成肽模仿靶蛋白中的特定序列，然后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项最新进展实现的：1。在噬菌体T7中建立了一个表达系统，该系统将主要衣壳蛋白的编码序列与大约150个碱基对的cDNA插入片段融合在一起；半自动化系统的发展，其中肽合成共价连接到一个96“针”的支持，大大简化了目标残基的高通量鉴定。该系统很容易扩展到384“引脚”或更大，并且可能完全自动化。该提案有两个步骤或目标：首先，完善克隆和表达最佳长度cdna的系统，并测试它们与特定蛋白质和合成肽目标相互作用的能力。其次，通过识别与髓磷脂结构蛋白相互作用的新分子来测试系统的多功能性，而髓磷脂结构蛋白的伴侣尚未被识别并验证其表达模式。我们选择髓磷脂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异常表型。因此，这些研究将首次允许在单个氨基酸取代与人类疾病状态中效应结合的丧失之间建立强有力的相关性。

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.