title: Expression, purification and functional reconstitution of FeoB, the ferrous iron transporter from Pseudomonas aeruginosa.
abstract: 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.
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK.
authors.forename: Saeed
authors.initials: S
authors.lastname: Seyedmohammad
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK.
authors.forename: Diana
authors.initials: D
authors.lastname: Born
authors.affiliation: School of Pharmacy & Medical Sciences, Sansom Institute for Health Research, University of South Australia, GPO Box 2471, Adelaide 5001, Australia. Electronic address: rietie.venter@unisa.edu.au.
authors.forename: Henrietta
authors.initials: H
authors.lastname: Venter
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D018441:Biofilms; D027682:Cation Transport Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D007501:Iron / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D009154:Mutation; D066329:Protein Aggregates / Q000187:drug effects; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000472:pathogenicity; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D027682:Cation Transport Proteins; D003902:Detergents; D008081:Liposomes; D066329:Protein Aggregates; D011994:Recombinant Proteins; D007501:Iron; D019204:GTP-Binding Proteins
keywords: Iron transporter; Membrane protein; Overexpression; Pseudomonas aeruginosa; Reconstitution