Abstract
Protein disulfide oxidoreductases (PDI) are redox enzymes that catalyze dithiol-disulfide exchange reactions in eukaryotic and bacterial cells. Several proteins belonging to this superfamily have been identified and characterized; thioredoxin, glutaredoxin, protein disulfideisomerase (PDI), disulfide bond forming (DsbA), and their homologs in prokaryotes are the most extensively studied members of this group. A highly thermostable protein disulfide oxidoreductase was first isolated from Sulfolobus solfataricus. From its ability to catalyze the reduction of insulin disulfides in the presence of dithiothreitol (DTT), the protein was considered a thioredoxin. The protein showed an unusually high molecular mass of 25 kDa and from amino acid composition analysis contained four cysteine residues. A homologous protein was subsequently purified from Pyrococcusfuriosus. More recently, the protein was crystallized, and its three-dimensional (3-D) structure at high resolution revealed structural details suggesting it may be related to the multidomain eukaryotic PDI. For this reason, the protein was more correctly named “protein disulfide oxidoreductase” from P furiosus ( Pf PDO). This chapter describes the purification of the Pf PDO, the cloning, and its gene expression in E. coli .
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