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

COBRE：路易斯维尔 RES FOUND INC：核心 D：NMR

• 批准号: 7959806
• 负责人: ANDREW N. LANE
• 金额: $ 6.03万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7959806
• 关键词: Affinity Chromatography; Apoptotic; Bacteria; Biological; Biophysics; Calmodulin; Cells; Centers of Research Excellence; Computer Retrieval of Information on Scientific Projects Database; Core Facility; Crystallography; Cytosine; DNMT3B gene; Data Collection; Differentiation and Growth; Ensure; Enzymes; Escherichia coli; Fibroblasts; Funding; Gene Expression Profile; Goals; Grant; Human; Inclusion Bodies; Insecta; Institution; Jurkat Cells; Label; Laboratories; Large T Antigen; Learning; Ligand Binding; Manuscripts; Metabolic; Metabolic Pathway; Metabolism; Methods; Methyltransferase; Mitochondria; Molecular Target; Peptides; Phenotype; Preparation; 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; 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. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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) will 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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 Andrew Lane，核心D总监 核心的主要目的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，并以高产率生产适合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。 该计划的资深研究人员和新员工正在意识到核心设施对他们研究的好处。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，以及广泛使用Core的代谢组学功能（见下文）。正在努力确保新征聘人员充分了解核心设施，并将积极鼓励他们利用独特的能力。 Chi Li博士（项目6）将使用代谢组学方法来了解凋亡蛋白在分离线粒体上的代谢结果。 Geoffrey Clark博士（项目7）正在使用蛋白表达核心生产人DNMT 3b（DNA（胞嘧啶-5-）-甲基转移酶3 β）作为GST融合物，用于表征和生物学实验。 Magda Kucia博士（项目10）正在研究用于再生治疗的干细胞。除了表征这些细胞在扩增过程中的基因表达谱外，她还对确定这些细胞的代谢表型感兴趣，以了解在能量和合成代谢方面生长和分化的精确要求。 Hong Ye博士（COBRE毕业生）利用表达核心设施进行蛋白质生产，咨询和分析，并将需要NMR功能对她通过X射线衍射方法解决的蛋白质结构进行一些功能分析。 Jason Chesney博士（COBRE毕业生）继续广泛使用该设施，并一直在分析iPFK 2和ras表达的代谢后果（以及RNAi敲除的影响）以及通过hTERT和大T抗原表达在许多细胞类型中永生化的影响，包括Jurkat细胞，MCF-7，支气管上皮细胞和成纤维细胞。使用NMR设备的代谢产物谱和同位素异构体分析有助于发现转化道路上细胞扰动的后果（有时具有令人惊讶的结果），以及验证（或以其他方式）潜在的抗癌靶酶。其中一些工作现已出版，其他手稿正在编写中。 Binks Wattenberg博士（COBRE毕业生）正在昆虫细胞和细菌中使用瞬时表达来表达鞘氨醇激酶（E。大肠杆菌）。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hexahis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NMR分析。此外，他一直在使用NMR设备分析靶向肽的溶液性质。