Submitted to: Protein Expression and Purification
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/10/2012
Publication Date: 4/1/2012
Citation: Vrentas, C.E., Onstot, S., Nicholson, E.M. 2012. A comparative analysis of rapid methods for purification and refolding of recombinant bovine prion protein. Protein Expression and Purification. 82(2):380-388.
Interpretive Summary: Bacterial production of a mammalian recombinant prion protein (rPrP) is a frequently used model system for the study of the chemical and physiological properties of the normal and disease causing forms of prion proteins. When bacteria produce these proteins they are not made in their natural shape or form, and require various subsequent laboratory steps to properly purify and fold the bacterially produced rPrP into a shape resembling its natural form. Our research here provides an improved method to produce rPrP for research purposes that is more efficient and enables our research to proceed at a faster pace than using previously published methods. The approach we found produces highly pure rPrP with a minimum of purification steps and a high yield per liter of induced bacterial culture is desired. We rigorously and directly compared several purification approaches for several key features of the purified protein such as purity, yield, and oxidation status. Recommendations are provided for the use of these methods in future work. These findings are applicable to any high-throughput recombinant prion studies and will inform researchers as to the utility of the purification approaches currently in the literature with regard to yield, purity, and other features of interest to study of rPrP.
Technical 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, overexpressed rPrP from the inclusion body form 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 rigorously and directly compare purification approaches for untagged bovine rPrP as adapted to rapid, "benchtop" formats useful for higher-throughput studies. An analysis of protein yield, purity, oxidation, and protein refolding revealed significant differences between preparative methods as adapted to the benchtop 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 the level of rPrP binding to cobalt affinity resin.