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: 7720765
• 负责人: ANDREW N. LANE
• 金额: $ 11.77万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7720765
• 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; Glutathione S-Transferase; 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; Numbers; 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; 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 seeing 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.

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