Pfu purification

Grow cells, induce with IPTG, spin down cells.

To 50 g cell paste add 200 ml of Buffer A:

50 mM Tris-HCl, 500 mM NaCl, pH 8.0, 1 mM beta mercaptoethanol

Lyse by freeze and thaw

Incubate at 70°C for 1 hour

Cool on ice for 15 min

Homogenize

Spin at 25,000 rpm for 30 min.

Load supernatant to a Ni column, pre-equilibrated with Buffer A , 500 mM NaCl; 50 mM Tris-HCl (pH 7,5), 20 mM Imidazole

Wash column with  Buffer A , 500 mM NaCl; 50 mM Tris-HCl (pH 7,5), 20 mM Imidazole

Wash column once 1 CV with Buffer C: 50 mM Tris-HCl (pH 7,5)

Elute protein with Buffer B: 50 mM NaCl; 50 mM Tris-HCl (pH 7,5), 150 mM Imidazole

Dilute eluted enzyme with  Buffer D: 50 mM Tris-HCl, pH 8.0 

Load onto Q-sepharose  HP column. Wash with Buffer D: 50 mM Tris-HCl, pH 8.0.

Linear gradient from  Buffer D to Buffer E: 50 mM Tris-HCl, pH 8.0, 600 mM NaCl to elute Pfu.

Collect and pool Pfu fractions.

Dilute combined enzyme with Buffer D: 50 mM Tris-HCl, pH 8.0.

Load diluted enzyme to a phosphocellulose or Heparin column (2.5×40 cm)  or at room temperature. Wash with Buffer D until the optical density measured at an absorbance of 280 nm drop  to baseline.

Elute Pfu with linear gradient Buffer D to  Buffer F: 1M  KCl; 50 mM Tris-HCl, pH 8,0.

Combine Pfu fractions

Run SDS-PAGE to check purity.