COBRE: LOUISVILLE RES FOUND INC: CORE D: NMR & PROTEIN PURIFICATION FACILITIES

COBRE：路易斯维尔资源公司：CORE D：NMR

• 批准号: 8360666
• 负责人: ANDREW N. LANE
• 金额: $ 12.3万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8360666
• 关键词: Affinity Chromatography; Apoptotic; Bacteria; Biological; Biophysics; Calmodulin; Cells; Centers of Research Excellence; Core Facility; Crystallography; DNMT3B gene; Data Analyses; Data Collection; Differentiation and Growth; Ensure; Enzymes; Escherichia coli; Fibroblasts; Funding; Gene Expression Profile; Goals; Grant; Human; Inclusion Bodies; Insecta; Jurkat Cells; Label; Laboratories; Large T Antigen; Learning; Ligand Binding; MCF7 cell; Manuscripts; Metabolic; Metabolic Pathway; Metabolism; Methods; Mitochondria; Molecular Target; National Center for Research Resources; Peptides; Phenotype; Preparation; Principal Investigator; Production; Property; Proteins; Publishing; RNA Interference; Recruitment Activity; Research; Research Infrastructure; Research Personnel; Resources; Scheme; Solutions; Source; Stable Isotope Labeling; Stem cells; Structure; Tissues; United States National Institutes of Health; Work; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; base; bronchial epithelium; cancer cell; cell type; cost; human DNMT3B protein; interest; metabolomics; programs; protein expression; protein purification; regenerative therapy; research study; sphingosine kinase

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Andrew Lane, Core D Director The primary aims of the core are to provide infrastructure for and support to structural analysis and metabolomics of cancer cells and tissue, and for producing proteins in high yield suitable for NMR analysis. This includes stable isotope labeling strategies including N-15 and C-13 precursors. Our goals continue to be to streamline both data collection and analysis as well as introduce new labeling strategies to provide more detailed information about specific metabolic pathways. Established researchers and new recruits to the program are realizing the benefits of the core facility for their research. This includes both protein production for biophysical analyses by X-ray crystallography, NMR and Biophysics, as well as extensive use of the metabolomics capabilities of the Core (see below). Efforts are being made to ensure that new recruits are fully cognizant of the core facilities and will be actively encouraged to use the unique capabilities. Dr. Chi Li (Project 6) is beginning to use metabolomics approaches for understanding metabolic consequences of apoptotic proteins on isolated mitochondria. Dr. Geoffrey Clark (Project 7) is using the Protein Expression core to produce human DNMT3b (DNA (cytosine-5-)-methyltransferase 3 beta) as a GST fusion that will be used for characterization and biological experiments. Dr. Magda Kucia (Project 10) is working on stem cells for regenerative therapy. In addition to characterizing the gene expression profile of such cells during expansion, she is interested in determining the metabolic phenotype of such cells to learn about the precise requirements for growth and differentiation in terms of energy and anabolic metabolism. Dr. Hong Ye (COBRE graduate) makes use of the expression core facilities for protein production, advice and analysis and will need the NMR capabilities for some functional analyses of the proteins whose structures she has solved by X-ray diffraction methods. Dr. Jason Chesney (COBRE graduate) has continued to make extensive use of the facility and has been analyzing the metabolic consequences of expression of iPFK2 and ras (and effects of RNAi knockdowns) as well as the effects of immortalization via hTERT and large T antigen expression in a number of cell types including Jurkat cells, MCF-7, bronchial epithelia and fibroblasts. Metabolite profiles and isotopomer analyses using the NMR facility have been instrumental in discovering consequences of cellular perturbations on the road to transformation (with sometimes surprising results), as well as validating (or otherwise) potential anticancer target enzymes. Some of this work has now been published and other manuscripts are in preparation. Dr. Binks Wattenberg (COBRE graduate) is expressing sphingosine kinase using transient expression in insect cells and also in bacteria (E. coli). The enzyme in the latter case enters inclusion bodies and has to be refolded after purification. The protein expression laboratory is working on purification schemes based on hexahis affinity chromatography, as well as calmodulin affinity chromatography, which has been shown to separate folded SK from unfolded enzyme. This will be used for biophysical studies, including crystallography, ligand binding, and NMR analyses. In addition, he has been analyzing the solution properties of targeting peptides using the NMR facility.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 Andrew Lane，核心 D 总监 该核心的主要目标是为癌细胞和组织的结构分析和代谢组学提供基础设施和支持，并以高产量生产适合 NMR 分析的蛋白质。这包括稳定同位素标记策略，包括 N-15 和 C-13 前体。我们的目标仍然是简化数据收集和分析，并引入新的标记策略以提供有关特定代谢途径的更详细信息。 该项目的老研究人员和新成员正在认识到核心设施对其研究的好处。这包括通过 X 射线晶体学、NMR 和生物物理学进行生物物理分析的蛋白质生产，以及 Core 代谢组学功能的广泛使用（见下文）。我们正在努力确保新员工充分了解核心设施，并积极鼓励他们使用独特的能力。 Chi Li 博士（项目 6）开始使用代谢组学方法来了解凋亡蛋白对分离线粒体的代谢影响。 Geoffrey Clark 博士（项目 7）正在使用蛋白质表达核心来生产人类 DNMT3b（DNA (胞嘧啶-5-)-甲基转移酶 3 beta）作为 GST 融合体，用于表征和生物实验。 Magda Kucia 博士（项目 10）正在研究用于再生疗法的干细胞。除了表征此类细胞在扩增过程中的基因表达谱外，她还对确定此类细胞的代谢表型感兴趣，以了解生长和分化在能量和合成代谢方面的精确要求。 Hong Ye 博士（COBRE 毕业生）利用表达核心设施进行蛋白质生产、建议和分析，并且需要 NMR 功能对她通过 X 射线衍射方法解析其结构的蛋白质进行一些功能分析。 Jason Chesney 博士（COBRE 毕业生）继续广泛使用该设施，并分析了 iPFK2 和 ras 表达的代谢后果（以及 RNAi 敲低的影响）以及通过 hTERT 和大 T 抗原实现永生化的影响在多种细胞类型中表达，包括 Jurkat 细胞、MCF-7、支气管上皮和成纤维细胞。使用 NMR 设备进行代谢物谱和同位素异构体分析有助于发现转化过程中细胞扰动的后果（有时会产生令人惊讶的结果），以及验证（或以其他方式）潜在的抗癌靶酶。其中部分作品现已出版，其他手稿正在准备中。 Binks Wattenberg 博士（COBRE 毕业生）正在利用昆虫细胞和细菌（大肠杆菌）中的瞬时表达来表达鞘氨醇激酶。后一种情况下的酶进入包涵体，纯化后必须重新折叠。蛋白质表达实验室正在研究基于六组氨酸亲和层析以及钙调蛋白亲和层析的纯化方案，该方案已被证明可以将折叠的 SK 与未折叠的酶分离。这将用于生物物理研究，包括晶体学、配体结合和核磁共振分析。此外，他还一直使用核磁共振设备分析靶向肽的溶液特性。