TY - CONF A1 - Vranic, Marija A1 - Starke, I. A1 - Weller, Michael G. A1 - Möller, H. M. T1 - Expression, purification and characterization of the recombinant cysteine-rich biomarker Hepcidin-25 N2 - Hepcidin regulates iron homeostasis in response to inflammation, erythropoietic demand, and iron stores. The native state of hepcidin-25 is an attractive target for the development of a reliable analytical tool that can quantify the hepcidin concentration in biological samples and reveal iron metabolic disorders. Therefore, a selective immunoassay would have to discriminate between different types of hepcidin and quantify only hepcidin-25’s concentration. The peptide contains a well-defined β-sheets and a β-hairpin loop stabilized by four disulfide bonds. Recently, it was shown that hepcidin-25 contains an ATCUN motif at its N-terminus. This motif is known to have high affinity towards Cu2+ and Ni2+. One of the aims of this study is to determine the three-dimensional (3D) structure of metal-bound hepcidin-25. Here, we present an optimized procedure for preparing natively folded hepcidin 25 (~2.80 kDa) and structural analysis of metal binding to hepcidin-25. Hepcidin was expressed as a His6-SUMO-hepcidin-25 fusion protein (~16.20 kDa) in Escherichia coli, Origami B strains, and purified as a soluble recombinant protein in three steps. After purification based on the nickel affinity chromatography, the purified His6-SUMO-hepcidin 25 fusion protein was cleaved by the SUMO-specific ULP1 protease. The liberated hepcidin 25 was further purified on a Superdex 30 16/600 column and folded in the last step of purification in the presence of glutathione. Freshly expressed hepcidin was kept in its reduced form to prevent misfolding and allow for efficient removal of the SUMO tag. The presence of natively folded hepcidin 25 after RP-HPLC was confirmed by ESI-MS and NMR spectroscopy. Based on published chemical shifts, we achieved a nearly complete assignment of the labeled and unlabeled hepcidin-25 at pH=3. Comparison of 1H chemical shifts and TOCSY spectra at pH=7 in the presence and absence of Ni2+ demonstrates that the metal binds at the N-terminus of hepcidin 25. Chemical shift changes due to metal complexation decrease further away from the metal binding site. T2 - 28th International Conference on Magnetic Resonance in Biological Systems, ICMRBS CY - Dublin, Irland DA - 19.08.2018 KW - Peptides KW - Metalloproteins KW - Copper KW - ATCUN KW - Nickel KW - NMR KW - SUMO KW - Fusion protein KW - Iron disorders KW - ESI-MS KW - RP-HPLC PY - 2018 AN - OPUS4-46944 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -