A general protocol for the expression and purification of the intact transmembrane transporter FeoB

Ferrous iron (Fe2+) transport is an essential process that supports the growth, intracellular survival, and virulence of several drug-resistant pathogens, and the ferrous iron transport (Feo) system is the most important and widespread protein complex that mediates Fe2+ transport in these organisms. The Feo system canonically comprises three proteins (FeoA/B/C). FeoA and FeoC are both small, accessory proteins localized to the cytoplasm, and their roles in the Fe2+ transport process have been of great debate. FeoB is the only wholly-conserved component of the Feo system and serves as the inner membrane-embedded Fe2+ transporter with a soluble Gprotein-like N-terminal domain. In vivo studies have underscored the importance of Feo during infection, emphasizing the need to better understand Feo-mediated Fe2+ uptake, although a paucity of research exists on intact FeoB. To surmount this problem, we designed an overproduction and purification system that can be applied generally to a suite of intact FeoBs from several organisms. Importantly, we noted that FeoB is extremely sensitive to excess salt while in the membrane of a recombinant host, and we designed a workflow to circumvent this issue. We also demonstrated effective protein extraction from the lipid bilayer through small-scale solubilization studies. We then applied this approach to the large-scale purifications of Escherichia coli and Pseudomonas aeruginosa FeoBs to high purity and homogeneity. Lastly, we show that our protocol can be generally applied to various FeoB proteins. Thus, this workflow allows for isolation of suitable quantities of FeoB for future biochemical and biophysical characterization.

Automated summary

In this study, an overproduction and purification system was developed to isolate sufficient quantities of FeoB protein from various organisms. The effectiveness of protein extraction from the lipid bilayer was demonstrated through small-scale solubilization studies. This approach was then successfully applied to the large-scale purifications of Escherichia coli and Pseudomonas aeruginosa FeoBs to achieve high purity and homogeneity.