Expression and purification of functionally active ferrous iron transporter FeoB from Klebsiella pneumoniae

The acquisition of ferrous iron (Fe²⁺) is an important virulence factor utilized by several hospital-acquired (nosocomial) pathogens such as Klebsiella pneumoniae to establish infection within human hosts. Virtually all bacteria use the ferrous iron transport system (Feo) to acquire ferrous iron from their environments, which are often biological niches that stabilize Fe²⁺ relative to Fe³⁺. However, the details of this process remain poorly understood, likely owing to the few expression and purification systems capable of supplying sufficient quantities of the chief component of the Feo system, the integral membrane GTPase FeoB. This bottleneck has undoubtedly hampered efforts to understand this system in order to target it for therapeutic intervention. In this study, we describe the expression, solubilization, and purification of the Fe²⁺ transporter from K. pneumoniae, KpFeoB. We show that this protein may be heterologously overexpressed in Escherichia coli as the host organism. After testing several different commercially-available detergents, we have developed a solubilization and purification protocol that produces milligram quantities of KpFeoB with sufficient purity for enzymatic and biophysical analyses. Importantly, we demonstrate that KpFeoB displays robust GTP hydrolysis activity (k_cat^GTP of ∼10^-1 s^-1) in the absence of any additional stimulatory factors. Our findings suggest that K. pneumoniae may be capable of using its Feo system to drive Fe²⁺ import in an active manner.