Structure, Mechanism and Interactions of Type IA Topoisomerases

项目摘要PI：玉清谢静 此设备补充申请申请资金购买Akta FPLC蛋白质 大规模纯化重组拓扑异构酶蛋白的纯化系统 是通过X射线结晶学获得拓扑异构酶的新结构所必需的，以及 蛋白质-蛋白质拓扑异构酶复合体构象的生物物理研究 PI的NIGMS主动赠款目标下的相互作用和配体相互作用动力学 R01-GM054226和R35-GM139817。AKTA FPLC蛋白质纯化系统 PI的实验室中提供的产品需要频繁维修，并需要更换，以避免大量 停机时间。更换Akta FPLC净化系统将使我们能够生产 快速高效，没有瓶颈毫克量的野生型和突变型 大肠杆菌和结核分枝杆菌拓扑异构酶I，大肠杆菌拓扑异构酶III，以及 人拓扑异构酶IIIB对DNA和DNA作用的结构和生物物理研究 RNA底物，以及抑制剂。这些蛋白质需要以高纯度生产，以便 我们在FIU的实验室进行的研究，也是我们在Argonne National Lab和 NIH用于结晶筛选和单分子研究。

Project Summary PI: Yuk-Ching Tse-Dinh This equipment supplement application requests funds to purchase an AKTA FPLC protein purification system for the large scale purification of recombinant topoisomerase proteins that are needed for obtaining new structures of topoisomerases by X-ray crystallography, and biophysical studies to elucidate topoisomerase complex conformations, protein-protein interactions and ligand interaction dynamics under the aims of the PI’s NIGMS active grants R01-GM054226 and R35-GM139817. The AKTA FPLC protein purification system currently available in the PI’s lab requires frequent repairs and needs to be replaced to avoid extensive down times. The replacement of the AKTA FPLC purification system would allow us to produce quickly and efficiently without bottlenecks milligram quantities of wild-type and mutant forms of Escherichia coli and Mycobacterium tuberculosis topoisomerase I, E. coli topoisomerase III, and human topoisomerase IIIB for structural and biophysical studies of their action on both DNA and RNA substrates, as well as inhibitors. The proteins need to be produced in high purity for studies in our lab at FIU, and are also needed by our collaborators at Argonne National Lab and NIH for crystallization screens and single-molecule studies.