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

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 安德鲁·莱恩，Core D总监 CORE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，并生产适合核磁共振分析的高产率蛋白质。这包括稳定的同位素标记战略，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，以提供关于特定代谢途径的更详细信息。 现有的研究人员和该项目的新成员正在意识到核心设施对他们的研究的好处。这包括用于通过X射线结晶学、核磁共振和生物物理学进行生物物理分析的蛋白质生产，以及广泛使用核心的代谢组学能力(见下文)。正在努力确保新征聘人员充分认识到核心设施，并将积极鼓励他们使用独特的能力。 池莉博士(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。 杰弗里·克拉克博士(项目7)正在利用蛋白质表达核心生产人类Dnmt3b(DNA(胞嘧啶-5-)-甲基转移酶3β)作为GST融合，将用于鉴定和生物学实验。 Magda Kucia博士(项目10)正在研究用于再生治疗的干细胞。除了确定这类细胞在扩增过程中的基因表达谱外，她还对确定这类细胞的代谢表型感兴趣，以了解在能量和合成代谢方面对生长和分化的准确要求。 Hong Ye博士(Cobre毕业生)利用Expression核心设施进行蛋白质生产、建议和分析，并将需要核磁共振功能来对她通过X射线衍射方法解决其结构的蛋白质进行一些功能分析。 Jason Chesney博士(Cobre毕业生)一直在继续广泛使用该设备，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实现永生化的影响，以及在许多类型的细胞中表达大T抗原的影响，包括Jurkat细胞、MCF-7、支气管上皮和成纤维细胞。利用核磁共振设施进行的代谢物图谱和同位素分析有助于发现细胞扰动在转化过程中的后果(有时会有令人惊讶的结果)，以及验证(或其他)潜在的抗癌靶酶。其中一些工作现已出版，其他手稿正在准备中。 Binks Wtenberg博士(Cobre毕业生)正在利用在昆虫细胞和细菌(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，必须在纯化后重新折叠。蛋白质表达实验室正在研究基于六丝亲和层析和钙调素亲和层析的纯化方案，这种层析已被证明可以将折叠的SK从未折叠的酶中分离出来。这将用于生物物理研究，包括结晶学、配体结合和核磁共振分析。此外，他一直在使用核磁共振设备分析靶向多肽的溶液性质。

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.