DNA Sequences coding for hamster (residues 90–231 and 23–231; accession K02234), deer (residues 24–234; accession AF156185) and sheep (residues 25–234; accession AJ567988) rPrPc residues were amplified and ligated into the pET41 vector (EMD Biosciences) and sequences were verified. After transforming the plasmids into E. coli Rosetta cells (EMD Biosciences), we expressed the rPrPc using the Overnight Express Autoinduction system (EMD Biosciences). Cell pellets from 0.25 L cultures were then put through two liquid nitrogen freeze thaw cycles and further lysed with BugBuster master mix (EMD Biosciences) to isolate the inclusion bodies. Next, the inclusion bodies were washed twice with 0.1× BugBuster, pelleted by centrifugation, and frozen at −20°C for later use. Then they were denatured in 8 M guanidine-HCl at pH = 8.5 on a rotator for 50 min at room temperature. Following a 16,000×g spin for 5 min, the denatured protein was then bound to Ni-NTA Superflow resin (Qiagen) that had been equilibrated in denaturing buffer [100 mM sodium phosphate (pH 8.0), 10 mM tris, 6 M guanidine-HCl]. The resin was loaded into a column and, using an AKTA Explorer system (GE Healthcare), the denatured protein was refolded on the column using a linear gradient to refolding buffer [100 mM sodium phosphate (pH 8.0) and 10 mM tris] over 4.5 h at a flow rate of 0.75 mL/min. Next the protein was eluted with a linear gradient to elution buffer [100 mM sodium phosphate (pH 5.8), 10 mM tris, and 500 mM imidazole] at 2 mL/min over 45 min. The protein fractions were diluted 5–10-fold into dialysis buffer [10 mM phosphate (pH 5.8)], filtered with a 0.2 µm syringe filter, and dialyzed. The concentration of rPrPc was determined by measuring absorbance at 280 nm and purity was ≥99%, as estimated by SDS-PAGE (see Results ), immunoblotting (data not shown), and mass spectrometry (mass = 16,238 amu; data not shown) [31] (link)–[33] (link). rPrPc preparations were aliquotted and stored at 0.2–0.4 mg/mL and −80°C.
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