@article {38415,
	title = {An optimized system for expression and purification of secreted bacterial proteins},
	journal = {Protein Expression and PurificationProtein Expression and Purification},
	volume = {46},
	year = {2006},
	type = {10.1016/j.pep.2005.09.003},
	abstract = {In this report, we describe an optimized system for the efficient overexpression, purification, and refolding of secreted bacterial proteins. Candidate secreted proteins were produced recombinantly in Escherichia coli as Tobacco Etch Virus protease-cleavable hexahistidine-c-myc eptiope fusion proteins. Without regard to their initial solubility, recombinant fusion proteins were extracted from whole cells with guanidium chloride, purified under denaturing conditions by immobilized metal affinity chromatography, and refolded by rapid dilution into a solution containing only Tris buffer and sodium chloride. Following concentration on the same resin under native conditions, each protein was eluted for further purification and/or characterization. Preliminary studies on a test set of 12 secreted proteins ranging in size from 13 to 130\&$\#$xa0;kDa yielded between 10 and 50\&$\#$xa0;mg of fusion protein per liter of induced culture at greater than 90\% purity, as judged by Coomassie-stained SDS{\textendash}PAGE. Of the nine proteins further purified, analytical gel filtration chromatography indicated that each was a monomer in solution and circular dichroism spectroscopy revealed that each had adopted a well-defined secondary structure. While there are many potential applications for this system, the results presented here suggest that it will be particularly useful for investigators employing structural approaches to understand protein function, as attested to by the crystal structures of three proteins purified using this methodology (B.V. Geisbrecht, B.Y. Hamaoka, B. Perman, A. Zemla, D.J. Leahy, J. Biol. Chem. 280 (2005) 17243{\textendash}17250).},
	keywords = {Pathogens, Secreted proteins, Toxins, Virulence factors},
	isbn = {1046-5928},
	author = {Geisbrecht, Brian V. and Bouyain, Samuel and M. Pop}
}
