Structure, Mechanism and Interactions of Type IA Topoisomerases

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.