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.