Recombinant Hamster Prion Protein Preparation

Recombinant hamster PrP proteins (residues 23–231 and 90–231; GenBank accession no. K02234) were prepared according to the method described previously [6 (link),7 (link)]. Briefly, the two DNA sequences of hamster PrP residues 23–231 and 90–231 were firstly ligated into the pRSET vector (cat. no. V35120; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and then the recombinant plasmids were transformed into BL21(DE3)pLysS competent cells (cat. no. C1500; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). After treated the bacteria pellets by ultrasonication, the lysates were denatured with guanidine-HCL and the expressed proteins were purified by chromatography using Ni-NTA Superflow resin (cat. no. 30,430; Qiagen, Hilden, Germany) in an XK 16/40 column (GE Healthcare Life Sciences, Little Chalfont, UK) at a flow rate of 2.3 ml per min. The purified proteins were dialysed into 10 mM sodium phosphate buffer (pH 5.8) and the concentrations of recombinant PrP proteins (rHaPrP23-231 and rHaPrP90-231) were adjusted to 500 µg/ml as determined by absorbance measured at 280 nm. Following filtration (0.22 µm syringe filter, cat. no. SLGP033RB, Merck millipore), the recombinant PrP proteins were aliquoted and stored at −80°C, respectively.