CORE--PROTEIN

核心--蛋白质

• 批准号: 7036470
• 负责人: Reinhard Jahn
• 金额: $ 2.75万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7036470
• 关键词: bacterial proteins; biomedical facility; gene expression; high performance liquid chromatography; light scattering; mass spectrometry; molecular chaperones; protein folding; protein purification; protein structure function; recombinant proteins; transfection /expression vector

Support is requested for Protein Production. While small-scale preparations of recombinant proteins using standard affinity purifications will be performed in the individual laboratories of the project, all projects require mg-amounts of highly purified recombinant and untagged proteins. The Core will provide technical and scientific support in two ways. First, it will optimize expression of proteins that either do not express at all or only at very low levels, including change of vectors, bacterial strains, co-expression of chaperones, or switching to an eukaryotic expression system. Second, the core will express and purify medium to large scale amounts of proteins using an array of conventional and affinity-based chromatographic methods. The core is equipped with a bacterial fermenter for large-scale expression, two state-of-the-art automated FPLC-systems, and a miniaturaized FPLC (SMART). An array of physico-chemical techniques will be used for initial characterization with respect to folding, oligomerization/aggregation and purity including CD-spectroscopy, multi-angle laser light scattering, and mass spectrometry. It is directd jointly by Drs. Fasshauer and Jahn, who will be assisted by M. Boeddener, an experienced Research Associate with several years of experience in all of the required techniques.

要求为蛋白质生产提供支持。虽然使用标准亲和纯化的重组蛋白的小规模制备将在项目的各个实验室进行，但所有项目都需要镁含量的高度纯化的重组蛋白和未标记的蛋白。该中心将以两种方式提供技术和科学支持。首先，它将优化那些完全不表达或仅在非常低水平表达的蛋白质的表达，包括改变载体、细菌菌株、伴侣蛋白的共表达，或切换到真核表达系统。第二，核心将表达和提纯中到大 使用一系列传统的和基于亲和力的层析方法来测量蛋白质的量。核心配备了一个用于大规模表达的细菌发酵罐，两个最先进的自动化FPLC系统，以及一个微型FPLC(SMART)。将使用一系列物理化学技术对折叠、齐聚/聚集和纯度进行初步表征，包括CD光谱、多角度激光光散射和质谱学。它由Fasshauer博士和Jahn共同执导，M.Boeddener将协助他们，M.Boeddener是一位经验丰富的研究助理，曾在 具备所有所需技术的经验。