Purification of Recombinant E. Coli Topoisomerase III for Structure-Based Drug Design Using Protein Crystallization

Type IA Topoisomerases are ubiquitous enzymes found throughout all life forms and species. These topoisomerases relieve the topographical constrains formed by DNA during processes like replication and transcription via a cleavage-religation mechanism performed through a catalytically active tyrosine residue in the primary structure of the enzyme. E. coli Topoisomerase III (EtopIII) is a type of Type IA topoisomerase, and its main function in the cell is as a decatenase, which means that it unlinks circular or intertwined pieces of genetic material and creates two unlinked segments of DNA from a singular linked chain. Structure-based determination of the enzyme’s three-dimensional structure via crystallization could be beneficial in finding a novel antibiotic drug that is able to inhibit this enzyme in vivo, a solution that is sorely needed in a world where antibiotic-resistant bacteria are an ever-growing problem. The crystallization of the enzyme can be undertaken by truncating a part of the C-terminal of the enzyme that makes it harder for it to crystallize and introduce a DNA/RNA ligand to observe its binding interactions with said substrate. For this to happen, the truncated recombinant mutant must be purified and concentrated to desirable levels for crystallization. The results of the experiment show that out of 4L of bacterial starting culture, a total of 34.57 mg of protein was able to be purified. This protein was then sent for crystallization experiments, of which results are still pending, but preliminary results indicate that the protein quickly aggregates in one of the RNA ligand conditions, which elicits a change in protocol for future purifications. Furthermore, results from a relaxation assay indicate that the protein exhibits some relaxation activity even after the truncation of the C-terminal chain. These results could expand the knowledge of this enzyme for future structure-based drug development.