title: Expression and Purification of the Individual Bam Components BamB-E.
abstract: BamB, BamC, BamD, and BamE are lipoproteins that, along with the integral membrane protein BamA, form the β-barrel assembly machinery (BAM) complex in the outer-membrane of Gram-negative bacteria. Elucidating the roles that these lipoproteins play in the β-barrel assembly process requires both structural and functional studies that rely on milligram quantities of pure protein. Here, we describe a simple protocol for expressing individual BamB-BamE proteins in Escherichia coli and purifying them by nickel affinity and size-exclusion chromatography. This protocol yields pure proteins in amounts that are sufficient for crystallization trials, in vitro protein-protein interaction studies, NMR, and other biochemical experiments.
authors.affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, South Science Building, 8888 University Drive, Burnaby, BC, Canada, V5A 1S6.
authors.forename: Suraaj
authors.initials: S
authors.lastname: Aulakh
authors.affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, South Science Building, 8888 University Drive, Burnaby, BC, Canada, V5A 1S6.
authors.forename: Kelly H
authors.initials: KH
authors.lastname: Kim
authors.affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, South Science Building, 8888 University Drive, Burnaby, BC, Canada, V5A 1S6. mpaetzel@sfu.ca.
authors.forename: Mark
authors.initials: M
authors.lastname: Paetzel
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D015870:Gene Expression; D009532:Nickel / Q000737:chemistry
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D009532:Nickel
keywords: BAM complex; BamB; BamC; BamD; BamE; Lipoprotein; Nickel affinity chromatography; Protein expression; Size-exclusion chromatography; β-barrel assembly machinery