CORE--PEPTIDE BIOCHEMISTRY

肽生物化学核心（核心B）具有两个主要目标：1）生成 并维持初级噬菌体展示文库作为所提出的 研究，2）设计和执行实验，以分离和表征 功能阻断肽对靶蛋白的作用 调查不同的战略将适用于每一个具体的 应用程序.对于已经克隆的蛋白质，重组 蛋白质将被纯化并固定在固体支持物上。这些包括 肝配蛋白-A1（以前的B61）与人IgG融合（肝配蛋白-A1/IgG）（项目1）， 从生产细胞系中免疫亲和纯化的Fas配体 EGF残基K652-A674与GST融合（项目5）。初级 将在重组体上选择肽噬菌体展示文库 proteins.选择的噬菌体将在靶上扩增和选择 蛋白质再次在这样的重复之后，肽编码序列从 能够特异性结合靶蛋白的噬菌体将融合到 GST或合成。它们将被用于测试阻断Eph激酶 信号转导（项目1）或Fas-Fas配体介导的细胞凋亡（项目2）。 在项目5中，肽序列本身将用于推断 与EGFR K652-A674相互作用的蛋白质。由于功能性重组 蛋白质不适用于离子通道ROMK 1（项目3），我们将 在Sf 9和CHO-Lec-1上表达。通过改变噬菌体对昆虫的选择， 过表达该通道的哺乳动物细胞的非特异性结合 与总的细胞淘选相关的影响将最小化。肽将 使用通道箝位系统合成和表征。

The Peptide Biochemistry Core (Core B) has two main goals: 1) to generate and maintain primary phage display libraries as tools for the proposed studies, 2) to design and execute experiments to isolate and characterize function-blocking peptides against the target proteins under investigation. Distinct strategies will be applied to each specific application. For proteins that have already been cloned, recombinant proteins will be purified and immobilized on solid supports. These include ephrin-A1 (formerly B61) fused to human IgG (ephrin-A1/IgG) (Project 1), Fas ligand that is immuno-affinity purified from a producing cell line (Project 2), and EGF residue K652-A674 fused to GST (Project 5). Primary peptide phage display libraries will be selected on the recombinant proteins. Selected phage will be amplified and selected on the target protein again. Following such repeated, peptide coding sequences from phage capable of specifically binding to target proteins will be fused to GST or synthesize. They will be used to test for blocking Eph kinase signaling (Project 1), or Fas-Fas ligand mediated apoptosis (Project 2). In project 5, the peptide sequences themselves will be used to deduce proteins that interact with EGFR K652-A674. Since functional recombinant proteins are not available for ion channel ROMK1 (Project 3), we will express it on Sf9 and CHO-Lec-1. By altering phage selection on insect and mammalian cells overexpressing the channel the non-specific binding associated with total cell panning will be minimized. Peptides will be synthesized and characterized using the channel clamping systems.