Effect of oxidized and reduced forms of Escherichia coli DsbC on protein refolding

DsbC, which catalyzes disulfide isomerization, was overproduced in the periplasm of Escherichia coli and purified from the periplasmic fraction by osmotic shock and anion-exchange chromatography. The active site of the purified DsbC was found to be an oxidized form (ox-DsbC) which could be converted to the reduced form (red-DsbC) by the addition of dithiothreitol. The effect of ox- and red-DsbC on the refolding of chemically denatured and reduced proteins with different numbers of disulfide bonds and free cysteine-thiol groups was investigated. Ox-DsbC facilitated the refolding of proteins with multiple disulfide bonds in both oxidative and reductive environments, while red-DsbC facilitated refolding only in the former. On the other hand, only red-DsbC facilitated the refolding of proteins with multiple free cysteine-thiol groups but either form of DsbC did not facilitate the refolding of proteins with only one cysteine-thiol group. It is therefore important to choose the form which suits the properties of the protein. Holo-chaperonin from Thermus thermophilus and DsbC demonstrated a synergistic effect on protein refolding.