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The selective permeability of the outer membrane (OM) of Gram-negative bacteria is mainly determined by the pore-forming proteins within it, which are called outer membrane proteins (OMPs). The proteinaceous apparatus required to fold and assemble the OMPs is known as the beta-barrel assembly machinery (BAM) complex. In Escherichia coli, the BAM complex is made up of five proteins. BamA is a beta-barrel protein integrated into the OM, while BamB, BamC, BamD and BamE are lipoproteins attached to the periplasmic side of the OM by a covalently bound lipid. Over-expression of BamA without its signal peptide results in insoluble inclusion body formation. I have investigated refolding strategies for E. coli BamA. The refolded BamA has been analysed by size-exclusive chromatography, heat-modifiability, circular dichroism spectroscopy and limited proteolysis. The experimental results are consistent with both a full length BamA and a truncated BamA construct being refolded properly using my discovered conditions. Both refolded constructs are able to crystallize. These initial crystallization conditions will be helpful in the structural analysis of E. coli BamA.
