Protein disulfide isomerase A3 (PDIA3) is a major member of the protein disulfide isomerase (PDI) family. PDI proteins commonly reside in the endoplasmic reticulum and mediate important thiol-disulfide interchanges during post-translational protein folding. Unlike other PDI family members, PDIA3 is ubiquitous in various organ systems. However, its physiological activity varies in other tissues. PDIA3 has been associated with cancer, airway inflammation, neurodegenerative diseases, and metabolic diseases. However, the mechanisms of the association of PDIA3 with these pathological conditions remain unclear. Recombinant PDIA3 (rPDIA3) is needed to clarify the interactions between PDIA3 and certain physiological phenomena. In the present study, we aimed to produce highly purified rPDIA3 for use in pathological experiments. We expressed rPDIA3 with a histidine-enriched elongated peptide tag in Escherichia coli and obtained rPDIA3 at 97.8% purity using consecutive His-tag and reverse-phase chromatography. Elongated peptide tags screened from artificially designated library had dual functions for protein expression and simple purification.