Purification of Vaccinia Virus Capping Enzyme.

The expression vector containing the coding sequences of the His-tagged D1 and D12 subunits of vaccinia virus capping enzyme was a kind gift from the Stephen Cusack laboratory, European Molecular Biology Laboratory, Grenoble, France (63). The coexpressed D1 and D12 proteins from the plasmid were purified from BL21 (DE3) pLysS cells (Life Technologies). Briefly, the cells were cultured in Terrific broth at 37 °C with 250 rpm shaking until the OD reached ∼1.0, followed by protein induction with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 20 °C overnight. The cells were harvested by centrifugation at 7,000 rpm at 4 °C for 20 min and resuspended in lysis buffer (20 mM Tris base, 200 mM NaCl, 20 mM imidazole, 10% glycerol, and 5 mM Tris[2-carboxyethyl]phosphine [TCEP], pH 8.0), and then lysed by freezing and thawing, followed by microfluidization. After centrifugation at 12,000 rpm at 4 °C for 30 min, the supernatant containing the His-tagged proteins was applied to an Ni-NTA affinity column (Qiagen) and eluted by elution buffer (20 mM Tris base, 200 mM NaCl, 250 mM imidazole, 10% glycerol, and 5 mM TCEP, pH 8.0). Proteins were dialyzed overnight at 4 °C in storage buffer (20 mM Tris⋅HCl, 100 mM NaCl, 10% glycerol, and 1 mM DTT, pH 8.0). The activity of the capping enzyme was assessed in a trial capping reaction using a 20-nt RNA. Capped RNA can be resolved from uncapped RNA on a 20% denaturing acrylamide gel (SequaGel; National Diagnostics) run at 220 V for 3 h. Only one species of capped RNA was generated by vaccinia virus capping enzyme with the cap moiety appended to the 5′ end of the in vitro transcribed RNA. The appropriate amount of capping enzyme was chosen to achieve near-100% capping efficiency.