3.1. Expression and purification of ORF8
As shown in Fig. 2A, the cell culture of E.coli induced by IPTG
(Lane 3) displayed a single band corresponding to ∼15 kDa, which
corresponded to ORF8. In contrast, this band was absent in the cell
culture without IPTG induction (Lane 2). This suggested that IPTG could
induce the expression of ORF8. Moreover, the band was present in the
inclusion body (Lane 4) and absent in the supernatant (Lane 5). Thus,
ORF8 was expressed by E.coli in the form of inclusion body.
The inclusion body was dissolved in the buffer containing 6 M guanidine
hydrochloride and loaded on a Ni Sepharose HP column (0.5 cm ×5 cm). The
column was eluted by the gradient imidazole. Two separated major peaks
were observed upon the imidazole elution (Fig. 2B). Peak 2 corresponding
to ORF8 was fractionated. ORF8 was in denatured and then renatured by
dropwise dilution of the solution.