Structural Investigation of FtsK and Close Homologs

Abstract

FtsK is an integral membrane DNA translocase of the Fts family found within Escherichia coli and with close homologs across many bacterial species. Some of those homologs (Tra family) are also involved in a variety of type IV secretion systems, commonly involved in bacterial conjugation and often in infection. Our work focused on the cloning, expression, purification, and crystallization of FtsK and several homologs, TrwB, TraD, and TraG, for the purpose solving the atomic resolution structure of these proteins. We prepared a variety of constructs in bacterial T7 based expression vectors for the purpose of expressing these proteins in E. coli. For the proteins that showed positive expression, the protein was solubilized with detergent and then purified through nickel-NTA affinity and size-exclusion chromatography (SEC). Results are shown through SEC reports and SDS-PAGE gels that indicate protein purity (by gel) and homogeneity and oligomeric state (by SEC). Many bacterial membrane based proteins, especially those of FtsK and its relatives are accesible targets for antibiotic intervention in humans as they are unique to bacterial systems. For this reason we hope that the procedures followed here may enable further development of our knowledge of these systems, that we might maintain a leg up on rapidly evolving pathogenic species.