CORE--PEPTIDE BIOCHEMISTRY

多肽生物化学核心(核心B)有两个主要目标：1)产生 并维护主要的噬菌体展示文库，作为建议的 研究，2)设计和执行实验以分离和表征 针对靶蛋白的功能阻断多肽 调查。不同的策略将应用于每个特定的 申请。对于已经克隆的蛋白质，重组 蛋白质将被提纯并固定在固体载体上。这些措施包括 与人免疫球蛋白融合的ephin-A1(前B61)(项目1)； 从生产细胞系中纯化的免疫亲和性Fas配体 (项目2)，和EGF残基K652-A674与GST融合(项目5)。主要 将多肽噬菌体展示文库选上重组 蛋白质。选定的噬菌体将被放大并在目标上进行选择 又是蛋白质。在这种重复的、来自于 能够与靶蛋白特异结合的噬菌体将与 商品及服务税或合成。它们将被用来测试是否阻断了EphK 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，肽序列本身将被用来推断 与EGFR K652-A674相互作用的蛋白质。自功能性重组 蛋白质不适用于离子通道ROMK1(项目3)，我们将 在Sf9和Cho-Lec-1上表达。通过改变昆虫和昆虫上的噬菌体选择 哺乳动物细胞过度表达通道的非特异性结合 与总的单元格平移相关联的值将最小化。多肽将是 利用通道钳制系统合成并表征了该化合物。

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.