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: 8167778
• 负责人: ANDREW N. LANE
• 金额: $ 12.42万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8167778
• 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; 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) 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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 核心D主任安德鲁·莱恩（Andrew Lane） 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，并支持高产的蛋白质，适合NMR分析。这包括稳定的同位素标记策略，包括N-15和C-13前体。我们的目标继续是简化数据收集和分析，并引入新的标签策略，以提供有关特定代谢途径的更详细信息。 该计划的知名研究人员和新的新兵正在意识到核心设施在研究中的好处。这既包括通过X射线晶体学，NMR和生物物理学的生物物理分析的蛋白质产生，以及核心代谢能力的广泛使用（见下文）。正在努力确保新的新兵完全认识到核心设施，并将积极鼓励使用独特的功能。 Chi Li博士（项目6）开始使用代谢组学方法来理解凋亡蛋白在分离的线粒体上的代谢后果。 杰弗里·克拉克（Geoffrey Clark）博士（项目7）正在使用蛋白质表达核心生产人DNMT3B（DNA（cytosine-5-） - 甲基转移酶3β）作为GST融合，将用于表征和生物学实验。 Magda Kucia博士（项目10）正在研究干细胞进行再生治疗。除了表征扩展过程中此类细胞的基因表达谱外，她还有兴趣确定此类细胞的代谢表型，以了解能量和合成代谢代谢方面生长和分化的确切需求。 Hong Ye博士（COBRE毕业生）利用了表达核心设施来生产蛋白质，建议和分析，并且需要NMR功能，以便对她通过X射线衍射方法求解的蛋白质的某些功能分析。 Jason Chesney博士（Cobre毕业生）继续广泛使用该设施，并一直在分析IPFK2和RAS表达的代谢后果（以及RNAi敲除的影响）以及通过HTERT和大型T抗原表达的不朽效果以及包括Jurkat细胞类型的大量jurkat Cells and Mcf-7，FronChial and bronChroina，BronChronChel and bronChroinch and broncheal and fromchel and tybro。使用NMR设施的代谢产物谱和同位素分析对发现转化道路上的细胞扰动的后果（有时令人惊讶的结果）以及验证（或其他）潜在的抗癌靶酶。现在已经出版了其中一些工作，其他手稿正在准备。 Binks Wattenberg博士（Cobre Gradure）正在使用昆虫细胞和细菌（大肠杆菌）中的瞬时表达表达鞘氨醇激酶。后一种情况中的酶进入包容体，必须在纯化后重新折叠。蛋白质表达实验室正在基于六个亲和力色谱法以及钙调蛋白亲和力色谱法进行纯化方案，已证明将其与展开的酶分开。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，他一直在分析使用NMR设施靶向肽的溶液特性。