2.3. Expression, purification and renaturation of ORF8
The strain (20 mL) was added in 1 L LB medium containing 100 μg/ml kalamycin and cultured for overnight with vigorous shaking at 37 °C. When the strain density was in the range of 0.6-0.8 at 600 nm, IPTG was added at a final concentration of 0.6 mM for incubation at 16℃ for 20 h. The cells were harvested and sonicated in an ice bath.
The inclusion bodies were solubilized by 50 mM Tris-HCl buffer (pH 8.0) containing 6 M guanidine hydrochloride and 10 mM reduced glutathione (buffer A). Then, the sample was loaded on a Ni Sepharose HP column (0.5 cm×5 cm, GE Healthcare, USA). The column was eluted with 50 ml buffer A and then with 50 ml buffer A containing 0.5 M imidazole. The fractions containing ORF8 were pooled and ORF8 was in denatured state. In order to renature ORF8, the pooled solution was added dropwise to a 50-fold excess of 50 mM Tris-HCl buffer (pH 8.0) containing 0.5 M L-arginine, 2 mM EDTA, 5 mM reduced glutathione, 0.5 mM oxidized glutathione and 0.5 M urea at 4°C. Finally, the renatured ORF8 was dialyzed against PBS buffer (pH 7.4) and used as the antigen.