Characterization of a metal‐dependent regulator protein from Thermobifida fusca

Iron dependent regulators are proteins found in a variety of pathogenic bacteria that cause diseases such as tuberculosis and diphtheria, but few such regulators have been thoroughly studied. The purpose of this research is to characterize a metal‐dependent regulator from Thermobifida fusca, the causative agent of farmer's lung. In comparison to homologous regulators, the regulator from T. fusca has a longer N‐terminal helix. Conformational changes have been shown to occur upon metal binding in related proteins, and it has been suggested that this helix is important in DNA‐binding. The target gene that encodes for the T. fusca regulator was cloned into the pET‐28b expression plasmid, and the protein was expressed in BL‐21(DE3) cells. The regulator was crudely purified using a nickel‐affinity column. Gel‐shift assays indicate the protein successfully binds the diphtheria and tuberculosis DNA consensus sequence in a metal‐dependent fashion. This research is supported by the Wabash College Byron K. Trippet and Haines Biochemistry Funds.