Editing T4 PNK Purification

Production and Purification of T4 Pnk—The wild-type and mutant<br>pET-PNK plasmids were transformed into E. coli BL21(DE3). Single<br>ampicillin-resistant colonies were inoculated into LB medium contain-<br>ing 0.1 mg/ml ampicillin, and 50-ml cultures were grown at 37 °C until<br>the A600 reached 0.5. The cultures were placed on ice for 30 min, then<br>adjusted to 0.3 mM isopropyl-␤-D-thiogalactopyranoside (IPTG), and<br>subsequently incubated at 17 °C for 15 h with continuous shaking. Cells<br>were harvested by centrifugation, and the pellets were stored at<br>Ϫ80 °C. All subsequent procedures were performed at 4 °C. Cell lysis<br>was achieved by treatment of thawed, resuspended cells with 1 mg/ml<br>lysozyme and 0.1% Triton X-100 in 5 ml of lysis buffer containing 50 mM<br>Tris-HCl (pH 7.5), 1.2 M NaCl, 15 mM imidazole, 10% glycerol, 1 mM<br>benzamidine, and 0.2 mM phenylmethylsulfonyl fluoride. The lysates<br>were sonicated to reduce viscosity, and insoluble material was removed<br>by centrifugation at 40,000 ϫ g for 20 min. The supernatants were<br>applied to 1-ml columns of Ni-NTA-agarose (Qiagen). The columns were<br>washed with 5 ml of lysis buffer and then with 2 ml of buffer A (50 mM<br>Tris-HCl (pH 7.5), 200 mM NaCl, 10% glycerol) containing 0.1 M imid-<br>azole. The columns were step-eluted with 0.3, 0.5, and 1.3 M imidazole<br>in buffer A<br><br>

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The Pnkp ORF was amplified by PCR with primers that introduced BamHI restriction sites<br>over the start codon and 3′ of the stop codon, thereby facilitating in-frame insertion into vector<br>pET28-His10-Smt3. A 2-liter culture of E. coli BL21(DE3)/pET28-His10-Smt3-Pnkp was<br>grown in Luria-Bertani medium containing 0.06 mg/ml kanamycin until the A600 reached 0.6;<br>the culture was then adjusted to 0.3 mM isopropyl β-D-thiogalactoside and incubated for 3.5<br>h at 30°C. Cells were harvested by centrifugation and the pellet was stored at −80°C. All<br>subsequent procedures were performed at 4°C. Thawed bacteria were resuspended in 125 ml<br>of lysis buffer (50 mM Tris-HCl, pH 7.5, 1.2 M NaCl, 15 mM imidazole, 10% glycerol).<br>Lysozyme, PMSF, benzamidine, and Triton X-100 were added to final concentrations of 1 mg/<br>ml, 0.2 mM, 1 mM and 0.2% respectively. The lysate was sonicated to reduce viscosity and<br>insoluble material was removed by centrifugation for 45 min at 17,000 rpm in a Sorvall SS34<br>rotor. The soluble extract (900 mg protein) was applied to a 12-ml column of Ni2+-<br>nitrilotriacetic acid-agarose (Qiagen). The column was washed sequentially with 50 ml of<br>buffer A (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 10% glycerol), 50 ml of 50 mM Imidazole<br>in buffer A, and 50 ml of 100 mM imidazole in buffer A. The bound His10-Smt3-Pnkp protein<br>was then eluted with 200 mM imidazole in buffer A. The elution profile was monitored by<br>SDS-PAGE. The peak Pnkp-containing fractions were pooled and dialyzed overnight against<br>50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM DTT, 10% glycerol. A faint cloudy precipitate<br>was removed from the dialysate by centrifugation. The clarified supernatant (81 mg protein)<br>was treated for 1 h at room temperature with 81 μg of the Smt3-specific protease Ulp1<br>(Mossessova and Lima, 2000). The digest was applied to a 4.5-ml column of Ni-agarose and<br>the tag-free Pnkp protein was recovered in the flow-through fractions.<br><br>