Copper chaperone for cytochrome c oxidase (Cox 17) is a 7 kDa copper-binding protein, which facilitates incorporation of copper ions into Cu-A site of cytochrome c oxidase. Cox 17 contains six conserved Cys residues and occurs in three different oxidative states, which display different metal-binding properties and stability. In the present study, we have elaborated technologies for production of partially oxidized human recombinant Cox17 in a bacterial expression system and purification of fully oxidized Cox17. For this purpose we used Escherichia coli Origami (TM) strain, which is deficient in thioredoxin and thioredoxin reductase systems and allows formation of disulfide bonds in cytoplasmic proteins. Fully oxidized Cox17 was purified by a simplified two-step procedure including gel filtration and cation exchange chromatography. By using mass spectrometry we demonstrated that application of 2-mercaptoethanol (2-ME) during purification leads to formation of its mixed disulfide adducts with Cox 17. Moreover, partially reduced Cox 17 can form mixed disulfide adducts also with the cellular reducing agent glutathione, which abolishes copper-binding ability of partially reduced Cox 17. (c) 2006 Elsevier Inc. All rights reserved.