Preparation of the Oxidized and Reduced Forms of Psoriasin (S100A7).

Abstract

Human S100A7 (psoriasin) is a metal-chelating host-defense protein expressed by epithelial cells. S100A7 possesses two Cys residues that generate two redox isoforms of the protein. In the oxidized form (S100A7ox), Cys47 and Cys96 form an intramolecular disulfide bond, whereas these residues exist as free thiols in the reduced form (S100A7red). In this chapter, we provide a step-by-step protocol for the purification of S100A7ox and S100A7red that affords each protein in high yield and purity. In this procedure, S100A7 is expressed in Escherichia coli BL21(DE3), and the homodimer is obtained following ammonium sulfate precipitation, folding, and column chromatography. Treatment of S100A7 with 1,4-dithiothreitol (DTT) affords S100A7red. A Cu(II)-catalyzed oxidation reaction is employed to obtain S100A7ox. A RP-HPLC method that allows for baseline separation of S100A7ox and S100A7red is provided, as well as a biochemical Zn(II)-binding assay that can be employed to evaluate the functional integrity of S100A7.