title: Expression, Purification, and Screening of BamE, a Component of the BAM Complex, for Structural Characterization.
abstract: In Gram-negative bacteria, integral outer membrane β-barrel proteins (OMP) are assembled by the β-barrel assembly machine complex, or BAM complex. This complex includes the essential components BamA, an OMP composed of a carboxyl terminal β-barrel domain and five polypeptide transport-associated domains (POTRA), and the lipoprotein BamD. In Escherichia coli, the complex contains an additional three lipoproteins, BamB, C and E required for efficient delivery of OMPs to the outer membrane. Here we provide methods for production, isotope labeling, purification, and functional screening of BamE for research purposes. Purification strategies of both the soluble and wild-type membrane-tethered forms of BamE are described using techniques including osmotic shock, Ni-NTA purification, and size-exclusion chromatography. Functional screening using a simple plate assay is also described which allows screening for defects in outer membrane permeability.
authors.affiliation: School of Cancer Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: Jeeves
authors.affiliation: School of Cancer Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
authors.forename: Pooja
authors.initials: P
authors.lastname: Sridhar
authors.affiliation: School of Cancer Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK. t.j.knowles@bham.ac.uk.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Knowles
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D007553:Isotope Labeling; D009154:Mutation; D009997:Osmotic Pressure; D012995:Solubility
chemical_list: D001425:Bacterial Outer Membrane Proteins; C523551:BamE protein, E coli; D029968:Escherichia coli Proteins
keywords: BAM complex; BamE; Beta-barrel membrane proteins; POTRA; Protein labeling; Structural biology