T4 PNK Purification

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

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