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.
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References
Voulhoux R, Bos MP, Geurtsen J et al (2003) Role of a highly conserved bacterial protein in outer membrane protein assembly. Science 299:262–265
Wu T, Malinverni J, Ruiz N et al (2005) Identification of a multicomponent complex required for outer membrane biogenesis in Escherichia coli. Cell 121:235–245
Bos MP, Robert V, Tommassen J (2007) Biogenesis of the Gram-negative bacterial outer membrane. Annu Rev Microbiol 61:191–214
Selkrig J, Leyton DL, Webb CT et al (2014) Assembly of β-barrel proteins into bacterial outer membranes. Biochim Biophys Acta 1848:1542–1550
Kim KH, Aulakh S, Paetzel M (2012) The bacterial outer membrane β-barrel assembly machinery. Protein Sci 21:751–768
Kim KH, Paetzel M (2011) Crystal structure of Escherichia coli BamB, a lipoprotein component of the β-barrel assembly machinery complex. J Mol Biol 406:667–678
Kim KH, Aulakh S, Tan W et al (2011) Crystallographic analysis of the C-terminal domain of the Escherichia coli lipoprotein BamC. Acta Crystallogr Sect F Struct Biol Cryst Commun 67:1350–1358
Kim KH, Aulakh S, Paetzel M (2011) Crystal structure of the β-barrel assembly machinery BamCD complex. J Biol Chem 286:39116–39121
Kim KH, Kang HS, Okon M et al (2011) Structural characterization of Escherichia coli BamE, a lipoprotein component of the β-barrel assembly machinery complex. Biochemistry 50:1081–1090
Albrecht R, Zeth K (2011) Structural basis of outer membrane protein biogenesis in bacteria. J Biol Chem 286:27792–27803
Heuck A, Schleiffer A, Clausen T (2011) Augmenting β-augmentation: structural basis of how BamB binds BamA and may support folding of outer membrane proteins. J Mol Biol 406:659–666
Noinaj N, Fairman JW, Buchanan SK (2011) The crystal structure of BamB suggests interactions with BamA and its role within the BAM complex. J Mol Biol 407:248–260
Knowles TJ, McClelland DM, Rajesh S et al (2009) Secondary structure and (1)H, (13)C and (15)N backbone resonance assignments of BamC, a component of the outer membrane protein assembly machinery in Escherichia coli. Biomol NMR Assign 3:203–206
Albrecht R, Zeth K (2010) Crystallization and preliminary X-ray data collection of the Escherichia coli lipoproteins BamC, BamD and BamE. Acta Crystallogr Sect F Struct Biol Cryst Commun 66:1586–1590
Warner LR, Varga K, Lange OF et al (2011) Structure of the BamC two-domain protein obtained by Rosetta with a limited NMR data set. J Mol Biol 411:83–95
Sandoval CM, Baker SL, Jansen K et al (2011) Crystal structure of BamD: an essential component of the β-barrel assembly machinery of Gram-negative bacteria. J Mol Biol 409:348–357
Knowles TJ, Sridhar P, Rajesh S et al (2010) Secondary structure and 1H, 13C and 15N resonance assignments of BamE, a component of the outer membrane protein assembly machinery in Escherichia coli. Biomol NMR Assign 4:179–181
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Aulakh, S., Kim, K.H., Paetzel, M. (2015). Expression and Purification of the Individual Bam Components BamB–E. In: Buchanan, S., Noinaj, N. (eds) The BAM Complex. Methods in Molecular Biology, vol 1329. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2871-2_14
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DOI: https://doi.org/10.1007/978-1-4939-2871-2_14
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2870-5
Online ISBN: 978-1-4939-2871-2
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