Cloning, expression, purification and characterization of an iron-dependent regulator protein from Thermobifida fusca

• We cloned the T. fusca gene encoding a putative IdeR into a pET expression vector.
• We purified the Tf-IdeR protein to greater than 93% purity.
• Tf-IdeR DNA binding is activated by divalent metal ions.
• Tf-IdeR binds the toxPO sequence from C. diphtheriae.
• Tf-IdeR binds the T. fusca EntS and ABC transport system cis-regulatory elements.

Iron-dependent regulators (IdeRs) control the transcription of a variety of genes associated with iron homeostasis in Gram-positive bacteria. In this study we report the cloning of a putative IdeR gene from the moderate thermophile Thermobifida fusca into the pET-21a(+) expression vector. The expressed protein, Tf-IdeR, was purified using immobilized metal affinity and size-exclusion chromatography, and yielded approximately 12–16 mg of protein per liter of culture. The purified Tf-IdeR protein binds the tox operator sequence in the presence of divalent metal ions. Two Tf-IdeR binding sites were identified in the T. fusca genome upstream of a putative enterobactin exporter and a putative ABC-type multidrug transporter.