Fluorescence methods for HT validation and production of protein complexes

用于 HT 验证和蛋白质复合物生产的荧光方法

• 批准号: 8454468
• 负责人: LAWRENCE S SHAPIRO
• 金额: $ 31.07万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8454468
• 关键词: Address; B-Lymphocytes; Binding; Bioinformatics; Biological Assay; Cancer Center; Carcinogenesis Mechanism; Cell physiology; Cells; Cistrons; Cleaved cell; Complex; Coupled; Data; Detection; Eukaryotic Cell; Fluorescence; Genes; Imagery; In Vitro; Label; Life; Link; Macromolecular Complexes; Mammalian Cell; Mediating; Medical; Methods; Molecular Sieve Chromatography; Monitor; Peptide Hydrolases; Process; Production; Proteins; Proteolysis; Proteomics; Reagent; Science; Scientist; Signal Pathway; Signal Transduction; Site; Structure; System; Testing; Time; Validation; Work; base; design; high throughput screening; interest; migration; new technology; polypeptide; protein complex; protein expression; protein protein interaction; public health relevance; reconstitution; screening; stable cell line; transcription factor; vector

DESCRIPTION (provided by applicant): Macromolecular complexes are of key importance in virtually all life processes. However, more powerful methods need to be developed to (1) validate potential interacting partners, (2) efficiently screen for complex formation, and (3) produce sufficient amounts of functional protein complexes for in depth functional and structural studies. To address these needs we will (1) develop new high-throughput fluorescence-based screens to assess protein-protein interactions; (2) develop rapid screening method to distinguish between transient and stably interacting partners. (3) Finally, since functional characterization and structure determination of protein complexes depends on the capability to produce them recombinantly in high yield, we will develop a multi-protein expression system, based on distinct fluorescent markers whose expression levels are correlated with each protein component. Together, these methods provide a high- throughput pipeline for validating, characterizing, and producing protein complexes for structural and functional studies. This platform, and the associated reagents developed under this proposal, will provide a much needed toolbox that will greatly aid any scientist studying eukaryotic protein complexes.

描述（由申请人提供）：在几乎所有生命过程中，大分子复合物至关重要。但是，需要开发更强大的方法以（1）验证潜在的相互作用伙伴，（2）有效筛选复合物的形成，（3）在深度功能和结构研究中产生足够量的功能蛋白质复合物。为了满足这些需求，我们将（1）开发新的高通量荧光筛选，以评估蛋白质 - 蛋白质相互作用； （2）开发快速筛选方法，以区分瞬态和稳定相互作用的伙伴。 （3）最后，由于蛋白质复合物的功能表征和结构测定取决于能够以高产量产生它们的能力，因此我们将基于与每个蛋白质成分相关的不同荧光标记的多蛋白表达系统。这些方法共同提供了一条高吞吐管道，用于验证，表征和生产结构和功能研究的蛋白质复合物。该平台以及根据该提案开发的相关试剂将提供急需的工具箱，该工具箱将极大地帮助任何研究真核蛋白质复合物的科学家。