Expression and purification of an iron‐dependent regulator protein from Thermobifida fusca

Iron dependent regulators are implicated in a variety of diseases caused by pathogenic bacteria such as tuberculosis and diphtheria, but few such regulators have been studied. The purpose of this research is to clone, express and purify the iron dependent regulator from Thermobifida fusca , the causative agent of farmer's lung. The target gene was inserted into a pET‐21a expression plasmid both with and without C‐terminal 6x‐His tags. These plasmids were then transformed into E. coli BL21 (DE3) cells for protein overexpression. Unlike the regulators from C. diphtheriae and M. tuberculosis , the non‐His‐tagged protein failed to bind a nickel‐affinity column under a variety of conditions. However, the 6x‐His‐tagged protein was purified using a nickel‐affinity column, yielding approximately 35 mg per liter of culture. Subsequent purification using a size‐exclusion column suggests this protein has a tendency to aggregate. Initial DNA‐binding studies indicate the protein successfully binds the diphtheria and tuberculosis DNA consensus sequence. This research was funded by the Wabash College Byron K. Trippet Fund and the Haines Biochemistry Fund.