Structure, Mechanism and Interactions of Type IA Topoisomerases

项目概要PI：Yuk-Ching Tse-Dinh 该设备补充申请要求资金购买AKTA FPLC蛋白 用于大规模纯化重组拓扑异构酶蛋白的纯化系统， 是通过X射线晶体学获得拓扑异构酶新结构所必需的， 生物物理研究，以阐明拓扑异构酶复合物构象，蛋白质-蛋白质 根据PI的NIGMS主动赠款的目标， R 01-GM 054226和R35-GM 139817。AKTA FPLC蛋白纯化系统目前 PI实验室提供的设备需要经常维修，需要更换，以避免大量的 停机时间。更换AKTA FPLC纯化系统， 快速和有效地没有瓶颈毫克量的野生型和突变形式的 大肠杆菌和结核分枝杆菌拓扑异构酶I、E.大肠杆菌拓扑异构酶III，和 人拓扑异构酶IIIB用于其对DNA和 RNA底物以及抑制剂。蛋白质需要以高纯度生产， 研究在我们的实验室在FIU，也需要我们的合作者在阿贡国家实验室和 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.