Journal
PROTEIN EXPRESSION AND PURIFICATION
Volume 101, Issue -, Pages 138-145Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2014.06.012
Keywords
Pseudomonas aeruginosa; Iron transporter; Membrane protein; Overexpression; Reconstitution
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Funding
- Royal Society [45605]
- University of South Australia
- Sansom Institute for Health Research
- BBSRC scholarship
- Erasmus Programme Scholarship
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The FeoB Fe(II) transporter from the drug resistant pathogen, Pseudomonas aeruginosa is essential for ferrous iron transport and is implicated in virulence and biofilm development. Hence it is an attractive target for the development of new anti-infective drugs. FeoB is an intriguing protein that consists of a cytosolic N-terminal GTPase domain and an integral membrane domain which most likely acts as ferrous iron permease. Characterisation of FeoB is critical for developing therapeutics aimed at inhibiting this protein. However, structural and functional analysis of FeoB is hampered by the lack of high yield homogenously pure protein which is monodisperse, stable and active in solution. Here we describe the optimised procedure for the recombinant expression of FeoB from P. aeruginosa and provide an evaluation of the most favourable purification, pH and detergent conditions. The functional reconstitution of FeoB in liposomes is also described. This represents the first detailed procedure for obtaining a pure, active and stable FeoB solution in milligram quantities which would be amenable to biochemical, biophysical and structural studies. (C) 2014 Elsevier Inc. All rights reserved.
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