Human peptide deformylase (hPDF), located in the mitochondria, has recently become a promising target for anti-cancer therapy. However, the expression of the hPDF gene in Escherichia coli is not efficient likely due to extremely high levels of CC content as well as the presence of rare codons. We performed codon optimization of the hPDF gene in order to reduce CC content and to eliminate rare codons. Putative stable secondary structures of the optimized gene were also reduced. Codon optimization increased the expression of hPDF protein (residues 63-243) presumably by reducing the CC content. A large amount of soluble hPDF was obtained upon its fusion with thioredoxin (Trx-hPDF), although an insoluble fraction was still dominant. We confirmed that Co2+ is an optimal metal for increasing the activity of purified Trx-hPDF, and that actinonin acts as an efficient inhibitor. Therefore, a large amount of purified hPDF protein would provide many benefits for the screening of various drug candidates. (C) 2009 Elsevier Inc. All rights reserved.