CORE--PEPTIDE BIOCHEMISTRY

核心--肽生物化学

• 批准号: 6651774
• 负责人: Bingcheng Wang
• 金额: $ 13.53万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6651774
• 关键词: biochemistry; biomedical facility; chimeric proteins; electroporation; immunoaffinity chromatography; peptide chemical synthesis; peptide library; peptide structure; protein purification; protein sequence; recombinant proteins; synthetic peptide

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.

肽生物化学核心（核心B）有两个主要目标：1）生成 并维护主要的噬菌体显示库作为提议的工具 研究，2）设计和执行实验以隔离和表征 功能阻断肽针对靶蛋白的肽 调查。不同的策略将应用于每个特定 应用。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载体上。这些包括 ephrin-A1（以前为B61）融合到人IgG（Ephrin-A1/IgG）（项目1）， 从产生的细胞系中纯化的免疫亲和力的FAS配体 （项目2）和EGF残基K652-A674融合了GST（项目5）。基本的 肽噬菌体显示库将在重组中选择 蛋白质。选定的噬菌体将被放大并在目标上选择 再次蛋白质。在这种重复之后，来自的肽编码序列 能够特异性结合靶蛋白的噬菌体将融合到 GST或合成。它们将用于测试阻断EPH激酶 信号传导（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。由于功能重组 蛋白质不适合Ion Channel Romk1（项目3），我们将 在SF9和CHO-LEC-1上表达它。通过改变对昆虫和 过表达通道非特异性结合的哺乳动物细胞 与总细胞池相关的将被最小化。肽会 使用通道夹具进行合成和表征。