Cox17, a copper chaperone for cytochrome c oxidase: expression, purification, and formation of mixed disulphide adducts with thiol reagents.
Copper chaperone for cytochrome c oxidase (Cox17) is a 7 kDa copper-binding protein, which facilitates incorporation of copper ions into Cu(A) site of cytochrome c oxidase. Cox17 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 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 Cox17. Moreover, partially reduced Cox17 can form mixed disulfide adducts also with the cellular reducing agent glutathione, which abolishes copper-binding ability of partially reduced Cox17.
Voronova
Anastassia
A
Institute of Gene Technology, Tallinn Technical University, Akadeemia tee 15, EE-12618 Tallinn, Estonia.
Kazantseva
Jekaterina
J
Tuuling
Marina
M
Sokolova
Niina
N
Sillard
Rannar
R
Palumaa
Peep
P
D000818:Animals; D001051:Apoenzymes / Q000302:isolation & purification; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003300:Copper / Q000737:chemistry*; D000079922:Copper Transport Proteins; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013439:Sulfhydryl Reagents / Q000737:chemistry; D013552:Swine
D001051:Apoenzymes; C491006:COX17 protein, human; D002352:Carrier Proteins; D000079922:Copper Transport Proteins; D004220:Disulfides; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D003300:Copper; D005978:Glutathione; D003545:Cysteine