Mature prion protein (PrP) is a 208-residue polypeptide that contains a single disulfide bond. We report an alternative method to purify recombinant mouse PrP produced in Escherichia coli. Bacterial inclusion bodies were solubilized in a buffer containing 2 M urea at pH 12.5. The solubilized protein was rapidly purified on a nickel affinity column without a chaotrope gradient, followed by ion-exchange chromatography. The yield and purity of PrP produced by this alternative approach was similar to that obtained using a conventional solubilization and on-column refolding protocol. Recombinant PrP produced using the non-reducing purification protocol is properly folded, as determined by circular dichroism, and a competent substrate for amyloid fibril formation, as determined by Thoflavin-T dye binding assays. In summary, this report describes a rapid method for producing properly folded recombinant PrP without reducing agents or a chaotrope gradient.