Editing Taq purification 2

Cells were harvested, resuspended in 2400 ml buffer A (50 mM Tris-HCl, pH 7.9,50 mM dextrose, 1 mM EDTA), collected by centrifugation, resus-<br>pended in 480 ml buffer A containing 4 mg/ml lysozyme (Sigma), and incubated at room temperature for 15 min. Buffer B (480 ml) was then added [ 10 mM Tris-HCl, pH 7.9, 50 mM KCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.5% Tween 20, 0.5% Nonidet P40) and the mixture was incubated in a 75°C water bath for 60 min in 180-ml aliquots to allow more rapid temperature equilibration. Cell debris and denatured protein was removed by centrifugation at 4°C for 15 min in a Sorvall GSA rotor at 8000 rpm. Subsequent operations were performed at 4 to 8°C. Taq DNA polymerase was recovered from this clarified lysate by precipitation with polyethyleneimine (PEI). Because the required PEI concentration was slightly variable and too much PEI inhibited precipitation, preliminary titrations were normally performed. Neutralized 10% PEI (Miles) was added to 200-~1 aliquots of lysate to a final concentration of 0.05 to 0.8% and the mixture was vortexed and incubated for 5 min at room temperature. The precipitate was recovered by spinning<br>for 5 min in a microcentrifuge and resuspended in 200 ~1 buffer C (20 mM HEPES, pH 7.9,l mM EDTA, 0.5 InM PMSF, 0.5% Tween 20,0.5% Nonident-P40) containing 0.25 M KC1 to elute the DNA polymerase. Residual precipitate was removed by recentrifugation and activity in dilutions of the supernatant<br>was determined by the primed Ml3 assay. After titration, neutralized 10% PEI was added dropwise to the remaining lysate to the optimum concentration<br>(usually 0.15%), incubated 10 min, and the precipitate collected by centrifugation for 20 min at 8000 rpm in a Sorvall GSA rotor. The pellet was washed once by resuspension in 240 ml buffer C containing 0.025 M KC1 and the precipitate was recovered by centrifugation. The Taq DNA polymerase was eluted from the precipitate by resuspension in 240 ml buffer C containing 0.15 M KC1 and the residual precipitate was removed by centrifugation. Resuspensions<br>of the PEI precipitates were routinely done with several strokes in a Dounce homogenizer with a loose pestle to ensure an even suspension. The PEI eluate was diluted to 50 mM KC1 with buffer C and applied to a 150-ml column of BioRex 70 ion exchanger (Bio-Rad) that had been preequilibrated<br>in buffer C containing 50 mM KCl. The column was then washed with 6 column volumes of the same buffer and the enzyme was eluted in a single step with&nbsp; buffer C containing 200 mM KCl. The peak of protein from this step was pooled and dialyzed for 12 h against two changes of 1 liter each storage buffer (20 mM HEPES, pH 7.9, 100 mM KCl, 0.1 InM EDTA, 0.5 InM PMSF, 1 mM dithiothreitol, 50% glycerol). No gelatin is added to this buffer (7) because the DNA polymerase is sufficiently concentrated for storage (lo-20 mg/ml). This preparation is stable for at least 10 min at 94”C, at least 24 h at room temperature,<br>and at least 6 rounds of freezing and thawing (data not shown). Aliquots of 100 ~1 are kept at -80°C except for working stocks, which are stable for at least several months when stored at -10°C.<br>

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The final BioRex 70 chromatography step removes slightly more extraneous protein, but more importantly removes residual PEI that can interfere with<br>DNA polymerase function. The final enzyme fraction still contains trace contamination from E. coli proteins (Fig. 1). Residual E. coli nucleic acids are undetectable by absorbance at 260 nm but can be detected by more sensitive assays at levels consistent with contaminations in commercial preparations<br><br><br>

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