CORE--PROTEIN

核心--蛋白质

• 批准号: 7596915
• 负责人: Reinhard Jahn
• 金额: $ 7.75万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7596915
• 关键词: Affinity; Affinity Chromatography; Arts; Chemicals; Individual; Laboratories; Lasers; Mass Spectrum Analysis; Methods; Molecular Chaperones; Postdoctoral Fellow; Preparation; Production; Proteins; Recombinant Proteins; Recombinants; Spectrum Analysis; Standards of Weights and Measures; System; Techniques; bacterial vector; base; experience; fast protein liquid chromatography; light scattering; protein expression

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.

要求提供蛋白质生产的支持。尽管将在项目的各个实验室中对重组蛋白进行小规模的重组蛋白制剂，但所有项目都需要高度纯化的重组和无标记蛋白的mg amounts。核心将通过两种方式提供技术和科学支持。首先，它将优化蛋白质的表达，该蛋白质根本不表达或仅在非常低的水平上表达，包括矢量的变化，细菌菌株，伴侣的共表达或切换到真核表达系统。其次，核心将表达并净化介质至大 使用一系列常规和基于亲和力的色谱方法的蛋白质量表。该核心配备了一个细菌发酵罐，用于大规模表达，两个最先进的自动FPLC系统和一个小型尿素化的FPLC（SMART）。一系列物理化学技术将用于有关折叠，寡聚/聚集和纯度的初始表征，包括CD光谱，多角度激光光散射和质谱。它是Drs共同指导的。 Fasshauer和Jahn将由M. Boeddener的协助 在所有必需的技术中经验。