Peroxiredoxins (Prxs), a family of thioredoxin-dependent peroxidases, are highly conserved in many organisms and function in detoxifying reactive oxygen species as well as other cellular processes. Six members of the Prx family are known in mammals, i.e., Prx-1 through -6. Among these proteins, only Prx-4 appears to contain a signal peptide that serves for localization in the endoplasmic reticulum, membrane translocation and secretion into the extracellular space, as demonstrated in a previous study using a baculovirus-insect cell system. The present study was conducted to determine whether the signal peptide-truncated mutant of rat Prx-4 is expressed as an enzymatically active form and is produced in large amounts. Two N-terminally truncated mutants were prepared by deletion of only the signal peptide and the larger region encompassing both the signal and the unique extension to Prx-4. These mutants were successfully produced within Spodoptera frugiperda 21 cells by infection with the recombinant baculoviruses, rather than by extracellular secretion. Both mutants were efficiently purified to homogeneity by two column chromatography steps. Biochemical characterization of the purified proteins showed that the truncated enzymes are enzymatically active and form an oligomeric structure, as reported for the mammalian Prx family. The findings also suggest that the unique extension plays a role in the regulation of non-covalent oligomerization. More than 4 mg of the purified proteins can be obtained from cells grown in monolayer cultures in twenty 75 cm(2) tissue culture flasks. The procedures described in this study permit recombinant Prx-4 to be prepared more efficiently and easily for purposes of crystallization and antibody preparation. (C) 2010 Published by Elsevier Inc.