title: A comparative analysis of rapid methods for purification and refolding of recombinant bovine prion protein.
abstract: 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.
authors.affiliation: Virus and Prion Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, 1920 Dayton Ave., Ames, IA 50010, USA.
authors.forename: Catherine E
authors.initials: CE
authors.lastname: Vrentas
authors.forename: Stephanie
authors.initials: S
authors.lastname: Onstot
authors.forename: Eric M
authors.initials: EM
authors.lastname: Nicholson
mesh_terms: D000818:Animals; D002021:Buffers; D002417:Cattle; D002846:Chromatography, Affinity; D003035:Cobalt / Q000737:chemistry; D002479:Inclusion Bodies / Q000737:chemistry; D011328:Prions / Q000737:chemistry / Q000302:isolation & purification*; D011485:Protein Binding; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D002021:Buffers; D011328:Prions; D011994:Recombinant Proteins; D003035:Cobalt