Bacterially-produced recombinant prion protein (rPrP) is a frequently used model system for the study of the properties of wild-type and mutant prion proteins by biochemical and biophysical approaches. A range of approaches have been developed for the purification and refolding of untagged rPrP expressed as inclusion bodies in Escherichia coli, including refolding by dialysis and simultaneous on-column purification and refolding. In order to perform a higher-throughput analysis of different rPrP proteins, an approach that produces highly pure rPrP with a minimum of purification steps and a high yield per liter of induced bacterial culture is desired. Here, we directly compare purification approaches for untagged bovine rPrP as adapted to rapid, small-scale formats useful for higher-throughput studies. An analysis of protein yield, purity, oxidation, and refolding revealed significant differences between preparative methods as adapted to the small-scale format, and based on these findings we provide recommendations for future purifications. We also describe the utility of a sensitive commercial kit for thiol analysis of these preparations, the pH dependence of dimer formation during refolding of bovine rPrP, and bovine rPrP binding to cobalt affinity resin. Published by Elsevier Inc.