Recombinant PrP production for RT-QuIC analysis

Truncated Bank voles PrP (BvPrP(90-231)) construct was purchased from GenScript. The construct was expressed in Escherichia coli BL21 (DE3) cells (Stratagene). Freshly transformed overnight culture was inoculated into Luria Bertani (LB) medium and 100 μg/mL ampicillin. At 0.8 OD600 expression was induced with isopropyl b-D galactopyranoside (IPTG) to a final concentration of 0.75 mM. Cells were grown in a BioStat-B plus fermentor (Sartorius). The cells were lysed by a homogenizer (PandaPLUS 2000) and the inclusion bodies were suspended in buffer containing 25 mM Tris-HCl, 5 mM EDTA, 0.8% TritonX100, pH 8, and then in bi-distilled water several times. Inclusion bodies containing BvPrP(90-231) were dissolved in 5 volumes of 8 M guanidine hydrochloride (GndHCl), loaded onto pre-equilibrated HiLoad 26/60 Superdex 200-pg column, and eluted in 25 mM Tris–HCl (pH 8), 5 mM ethylenediaminetetraacetic acid (EDTA), and 6 M GndHCl at a flow/rate of 2 mL/min. Proteins refolding was performed by dialysis against refolding buffer (20 mM sodium acetate and 0.005% NaN₃ (pH 5.5)) using a Spectrapor membrane. Purified protein was analyzed by SDS-polyacrylamide gel electrophoresis under reducing conditions and Western blot.