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，然而用于鉴定新的相互作用基因产物的方法是耗时的并且范围有限。 本提案的目标是开发一种方法，用于高通量筛选多个靶蛋白的结合伴侣，并快速鉴定它们结合的结构域。 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。分批分离携带直接结合靶蛋白的肽序列的噬菌体。 然后将模拟靶蛋白中特定序列的重叠合成肽用作噬菌体结合的靶标以鉴定特异性结合结构域。 这种新的范式是可能的两个最近的进展：1。在噬菌体T7中的表达系统的开发，其融合了主要衣壳蛋白的编码序列与约150个碱基对的cDNA插入物，和2。开发了一种半自动化系统，其中合成的肽共价连接到96“pin”支持物上，大大简化了靶残基的高通量鉴定。 该系统可以扩展到384个“pin”或更多，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，以及测试它们与定义的蛋白质和合成肽靶标相互作用的能力。第二，通过鉴定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，所述髓磷脂结构蛋白的伴侣尚未被鉴定，并且验证其表达模式。 我们选择髓鞘特异性蛋白质作为实验靶点，因为这些蛋白质的突变会导致严重的髓鞘形成障碍表型。 因此，这些研究将首次允许在单个氨基酸取代和人类疾病状态中效应物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.