Figure 2

Expression and purification of recombinant PrV vhs protein using E. coli . The coding region of PrV vhs was inserted downstream of the NUS gene in pET44 vector (A). In this expression system, the recombinant vhs protein was fused with NUS tag protein and a his tag at the N and C terminus, respectively. Under IPTG induction, NUS-vhs-his with a predicted molecular weight of ~110 kDa was expressed (B, lane 2) and then purified using Ni–NTA beads (lane 3). Subsequently, the N-terminal NUS tag was removed by thrombin treatment (lane 4). M protein marker; Lane 1 non-induction lysate. The identity of the PrV vhs was initially confirmed by Western blot analysis using antibodies against the his tag (C). The arrow indicates the uncleaved NUS-vhs-his recombinant protein, and the thin arrowheads indicate the NUS tag and vhs-his after thrombin digestion. The ribonuclease activity of PrV vhs was tested using single stranded RNA (D). RNA substrates generated by in vitro transcription were incubated with assay buffer (mock), NUS protein (as a negative control), or recombinant PrV vhs protein for the indicated times (0, 10, 20, 40, and 60 min). The RNA reaction products were then analyzed by 1.3% agarose-formaldehyde gel electrophoresis and the amount of RNA remaining was measured by imageJ system. The experiment was repeated three times and the RNA content at the time point of 0 h was set to 100% (E).