We have recently used two different techniques that appear to both work very efficiently to remove the contaminating DNA. The simplest technique is the use of Millipore 300,000 MW cutoff centrifuge filters (cat. no. UFC4TMK25), available from Sigma (cat. no. M-2411). The enzyme readily passes through the filter, even in the presence of 50% glycerol storage buffer, while the contaminating DNA is retained by the filter. Since it takes several hours of centrifugation for the glycerol-containing buffer to pass through the filter, a refrigerated centrifuge should be used (even though the enzyme is heat stable).
An alternative method to remove the contaminating DNA is the use of hydroxylapatite. Ten grams of hydroxylapatite is washed and equilibrated with 10 mM potassium phosphate (pH 7.5), 10 mM b-mercaptoethanol, 5% glycerol. Both the enzyme and DNA bind to hydroxylapatite in this buffer. After applying the lysate to the hydroxylapatite and allowing it to bind for 30 minutes, the hydroxylapatite is washed with ten ml of the same buffer (10 mM potassium phosphate, pH 7.5, 10 mM b-mercaptoethanol, 5% glycerol). The enzyme is then eluted using 0.5 M potassium phosphate (pH 7.5), 10 mM b-mercaptoethanol, 5% glycerol. The process can be accomplished in a batchwise manner to simplify the procedure, or can be performed in a column. The enzyme elutes from the column at approximately 0.2 M potassium phosphate if a gradient elution is performed rather than a batchwise process. Due to the very high concentration of the enzyme, subsequent dilution into storage buffer eliminates the need for dialysis.
Significant loss of enzymatic activity is frequently encountered during the dilution of the enzyme into storage buffer. This is why we recommend a two-step dilution process as described in the BioTechniques report, and also recommend performing both steps very slowly and gradually.