Viccinia Capping Enzyme Purification
The full-length capping enzyme heterodimer was obtained by co-expression of the D1 and D12 subunits cloned in tandem into pET16b (Novagen) as previously described (Luo et al., 1995). An N-terminal His10 tag, cleavable by tobacco etch virus (TEV) protease, was appended to the D1 subunit. After transformation into E. coli BL21Star(DE3) pLysS, cells were grown in LB medium containing ampicillin (10 μg ml−1) and chloramphenicol (34 μg ml−1) at 37oC to an OD600 of 0.7 and then induced with 0.5 mM IPTG and grown overnight at 20oC. The sub-complex encoding the D1CTD domain (residues 545–844) and D12 subunit was obtained by PCR amplification using the complete D1-D12 co-expression plasmid as template. Transformed E. coli BL21Star(DE3) cells were grown in LB medium containing ampicillin (10 μg ml−1) at 37oC to an OD600 of 0.7 and then induced with 0.5 mM IPTG at 28oC for 5-8 h. For both constructs, the thawed cell pellet was resuspended in 40 mM Tris-HCl buffer at pH 8.0, 200 mM NaCl, 40 mM imidazole and lysed by sonication in the presence of a protease inhibitor cocktail (Roche Diagnostics). The protein was purified with Ni-NTA resin (Qiagen) following the manufacturer’s instructions. After His-tag cleavage overnight at 4oC with His-tagged TEV protease, the protein extract was applied to a heparin column (Pharmacia) and eluted with a linear gradient of 100-800 mM NaCl. After a second round of Ni-NTA resin, the obtained protein complex was applied to a Superdex200 chromatography (Pharmacia) in 40 mM Tris-HCl buffer at pH 8.0, 200 mM NaCl and 5 mM DTT, with a typical yield per litre of culture of about 1 and 20 mg for the full-length and D1CTD-D12 sub-complex respectively. Seleno-methionine (SeMet) substituted sub-complex was produced using E. coli BL21Star(DE3) in a defined minimal medium containing 60 mg l-1 of SeMet (Acrós Organics). Purification of the SeMet-labelled sub-complex was done essentially as for the native protein.
pET-His6-D1-C plasmids encoding tagged wild-type and mutant catalytic subunits were transformed into E. coli BL21-CodonPlus(DE3) together with plasmid pET-D12 encoding the nontagged stimulatory subunit. Cultures (500 mL) derived from single transformants were grown at 37°C in LB medium containing 50 μg/mL kanamycin, 100 μg/mL ampicillin, and 50 μg/mL chloramphenicol until the A 600 reached ~0.6. The cultures were placed on ice for 30 min and then adjusted to 0.2 mM IPTG and 2% (v/v) ethanol, and incubation was continued for 20 h at 17°C with constant shaking. Cells were harvested by centrifugation and stored at −80°C. All subsequent procedures were performed at 4°C. Thawed bacteria were resuspended in 25 mL of buffer A (50 mM Tris-Hcl at pH 8.0, 150 mM NaCl, 10% glycerol). Phenylmethylsulfonyl fluoride and lysozyme were added to final concentrations of 500 μM and 100 μg/mL, respectively. After incubation on ice for 30 min, Triton X-100 was added to a final concentration of 0.1%, and the lysates were sonicated to reduce viscosity. Insoluble material was removed by centrifugation. The soluble extracts were mixed for 30 min with 2 mL of Ni2+-NTA-agarose (Qiagen) that had been equilibrated with buffer A containing 0.01% Triton X-100. The resins were recovered by centrifugation, resuspended in buffer A, and poured into columns. The columns were washed with 10 mL of 20 mM imidazole in buffer A and then eluted stepwise with 2.5 mL aliquots of buffer A containing 50, 100, 250, and 500 mM imidazole. The 250-mM imidazole eluates containing the recombinant vaccinia proteins were dialyzed against buffer B (50 mM Tris-Hcl at pH 8.0, 100 mM NaCl, 10% glycerol, 2 mM DTT, 2 mM EDTA, 0.01% Triton X-100) and then stored at −80°C.