Editing Pfusion DNA Polymerase Purification

Suspend 183 g of cells in 550 ml of buffer A (10 mM KPO4 buffer, pH 7.4; 1.0 mM EDTA, 1.0 mM beta-mercaptoethanol) containing 0.1 M NaCl Sonicate for 5 minutes at 4° C. 

Spin the lysate at 15,000 g for 30 minutes at 4° C. 

Load the supernatant solution to a 470 ml Affigel blue column (Biorad). 

Wash the column with 1000 ml of buffer A containing 0.1 M NaCl. 

Elute the column with a 2000 ml linear gradient from 0.1 to 2.0 M NaCl in buffer A. 

The DNA polymerase eluted as a single peak at approximately 1.3 M NaCl (contains 80% of the activity applied. 

Dialyse the peak activity of DNA polymerase (435 ml) against 4 liters of buffer A. 

Load to 80 ml Phosphocellulose column, equilibrated with buffer A containing 0.1 M NaCl. 

Wash the column with 160 ml of buffer A containing 0.1 M NaCl, 

Elute the enzyme activity with 1000 ml linear gradient of 0.1 to 1.0 M NaCl in buffer A. 

The activity eluted as a single peak at 0.6 M NaCl and represented 74% of the activity applied. 

Dialyse the pooled activity (150 ml) against 900 ml of buffer A 

Load to a 42 ml DNA-cellulose column. 

Wash the column with 84 ml of buffer A containing 0.1 M NaCl 

Elute the enzyme activity with a linear gradient of buffer A from 0.1 to 1.0 M NaCl. 

The DNA polymerase activity should be eluted as a single peak at 0.3 M NaCl, and represented 80% of the activity applied. 

Pool the activity (93 ml), dialyze against 2 liters of buffer A containing 0.05 M NaCl. 

Load to a 1.0 ml HPLC mono-Q column. 

Elute the DNA polymerase activity with a 100 ml linear gradient of 0.05 M to 1.0 M NaCl in buffer A. 

The DNA polymerase activity should be eluted as a single peak at 0.1 M NaCl. 

Pool the fractions (3.0 ml), dilute to 6 ml with buffer A 

Load to an 1.0 ml HPLC mono-S column (Pharmacia) 

Elute with a 100 ml linear gradient in buffer A from 0.05 to 1.0 M NaCl. 

The activity should be elute as a single peak at 0.19 M NaCl. 

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[[Category:Protocols]]