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

该子项目是利用 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来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 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。 该项目的资深研究人员和新招募人员都看到了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（见上文）。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）对使用代谢组学方法来理解分离线粒体上凋亡蛋白的代谢结果感兴趣。 Hong Ye博士（项目5）利用表达核心设施进行蛋白质生产、建议和分析，并需要核磁共振能力来对她通过X射线衍射方法解决了结构的蛋白质进行一些功能分析。 J. 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（包括Jurkat细胞，MCF-7，支气管上皮细胞和成纤维细胞）中永生化的影响。使用NMR设备的代谢物谱和同位素异构体分析一直是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（或以其他方式）潜在的抗癌靶酶的工具。其中一些工作现已出版，其他手稿正在编写中。 B医生。Wattenberg（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。coli）。在后一种情况下，酶进入包涵体，并且在纯化后必须重折叠。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。

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 above). 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 interested in using metabolomics approaches for understanding metabolic consequences of apoptotic proteins on isolated mitochondria. Dr. Hong Ye (Project 5) makes use of the expression core facilities for protein production, advice and analysis and will need the NMR capabilites for some functional analyses of the proteins whose structures she has solved by X-ray diffraction methods. Dr. J. 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 is 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. B. 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.