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. 2013 Feb 4;8(2):e55569. doi: 10.1371/journal.pone.0055569

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© 2013 Storm et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Chromatogram of size exclusion chromatography at denaturing conditions, separating full length His-FeCh with molecular mass of 47 kDa from truncation products and other contaminants. Inlet: 12% SDS-gel stained by PageBlue (Fermentas) of the elution peaks. Peak numbers in the chromatogram match lane numbers of the inlet. (B) Purification of soluble recombinant His-FeCh co-expressed with GroEL/GroES (lane 1) or His-FeChΔ347 with molecular mass of 41.4 kDa (lane 2). (C) Immunoblot using HRP-conjugated anti-His antibody (Qiagen) detecting the soluble target protein purified from E. coli. Lane 1: His-FeCh co-expressed with GroEL/GroES, lane 2: without co-expression with chaperones no recombinant His-FeCh can be immunodetected. (D) Native size exclusion chromatography on full-length FeCh, the monomer has a molecular weight of 44 kDa. Inlet in D: Activity traces of an isolated purely monomeric fraction (filled circles) and a mix of oligomeric states (open circles).