Purification of Recombinant Mouse Prion Protein

The plasmid pET-11a (Novagen) encoding for the full-length MoPrP(23–230) was kindly provided by Prof. J.R. Requena (University of Santiago de Compostela, Santiago de Compostela, Spain). An overnight culture of E. coli Rosetta 2 (DE3) (Novagen) freshly transformed with the plasmid was added at 37°C to 2 L of Zym-5052 medium [40] (link). Cells were grown in a 2 L fermenter system (Sartorius), harvested after 24 hours and lysed by homogenizer (Panda plus, GEA Niro Soavi). Inclusion bodies were washed and solubilized according to [41] (link). MoPrP was purified using its octapeptide repeat sequence as natural affinity tag for nickel. MoPrP was loaded onto a 5-mL HisTrap column (GE Healthcare) equilibrated in binding buffer (2 M GndHCl, 500 mM NaCl, 20 mM Tris, pH 8) and eluted with 500 mM imidazole. Subsequently, the protein was purified by reverse phase (Jupiter C4, Phenomenex) and separated using a gradient of 0–95% acetonitrile and 0.1% trifluoroacetic acid. The purified protein was lyophilized and refolded in acetate buffer (25 mM NaOAc, 0.005% NaN₃, pH 5.5) according to [41] (link).