Editing PEI Precipitation of Taq

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9. Taq DNA polymerase can be recovered from this clarified lysate by

*Precipitation with polyethyleneimine (PEI), Neutralized 10% PEI (Miles) at a final concentration of 0.05 to 0.8%, usually 0.15% or 0.6% (should be titrated using 200 ul aliquots). Precipitate can be resuspended in 200 ul '''Buffer C''' (20 mM HEPES, pH 7.9, 0.1 mM EDTA, 0.5% Tween 20,&nbsp; 0.25 M KCl to elute the DNA polymerase. After spining, the supernatant can be either mixed with solid ammonium sulfate to 50% saturation to precipitate Taq DNA polymerase,&nbsp; or directly used for next step purification.

'''Preparing Neutralized 5% (w/v) PEI Stock Solution''': One hundred milliliters of 50% (w/v) PEI is dissolved in 800 ml of deionized water. The solution is adjusted to pH 7.9 by adding concentrated HC1 (approximately 38-40 ml) with stirring.

*Precipitation with solid ammonium sulfate to 50% saturation to precipitate Taq DNA polymerase. The pellet can be dissolved in Ni washing buffer.

<s>10. While mixing using stirrer bar,&nbsp; slowly add solid&nbsp;dibasic potassium phosphate powder to the clarified lysate to a final concentration of 10%.</s>

<s>11. Add equal volume of ethanol to the 10% salt solution and mixed vigorously for several seconds, spin for 5 min at 6000 rpm.</s>

<s>12. Carefully remove the bottom aqueous phase containing nucleic acid. Collect and upper orgainc phase containing Taq DNA polymerase. Do not collect the interface containing denatured protein.</s>

<s>13. Load the orgainc phase to a Ni column. Wash column with Ni washing buffer. Elute Taq DNA polymerase using Buffer B containing 150 mM Imidazole.</s>
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When protein is eluted from the PEI pellet, PEI is also released into solution. If the eluate is dialyzed or diluted to lower ionic strength, the<br/>free protein and PEI will again interact and precipitate. Therefore, one must remove the free PEI. This is most commonly done by adding enough<br/>ammonium sulfate to the eluate to precipitate the protein. At this high ionic strength (often 40-60% saturated ammonium sulfate), PEI will not<br/>bind to protein and will remain in the supernatant after the ammonium sulfate precipitate is pelleted. This procedure removes the vast majority<br/>of the PEI and is sufficient for most proteins. If a protein is especially sensitive to residual traces of PEI, then a further step is required. The<br/>easiest method is to thoroughly resuspend the ammonium sulfate pellet in 40-60% saturated ammonium sulfate (sufficient to keep the protein<br/>insoluble) and recentrifuge. This way, any PEI physically trapped in the pellet will be removed.

Another method to remove PEI involves passing the eluate, or the dissolved ammonium sulfate precipitate, through a phosphocellulose column at 1 M NaCI. The protein will not bind to the column at this high ionic strength, but the PEI binds very tightly and is completely removed.

'''To further purify PEI-precipitated Taq Polymerase using BioRex 70:'''

1. Dilute the PEI eluate to 50 mM KCl or NaCl with '''Buffer D '''(20 mM HEPES, pH 7.9,&nbsp; 0.5% Tween 20)

2. Apply to a 150-ml column of BioRex 70 ion exchanger (Bio-Rad) that had been preequilibrated in '''Buffer E''' (20 mM HEPES, pH 7.9, 0.5% Tween 20, '''50''' mM KCl or NaCl).

3. Wash with 6 column volumes of '''Buffer E'''

4. Elute the enzyme with '''Buffer F''' (20 mM HEPES, pH 7.9,&nbsp; 0.1% Tween 20, '''200''' mM KCl).

5. Pool the fractions containing the peak of protein

6. Dialyzed for 12 h against two changes of 1 liter each '''Storage Buffer''' (20 mM HEPES, pH 7.9, 100 mM KCl, 0.1 mM EDTA, 0.5 mM PMSF, 1 mM dithiothreitol, 50% glycerol).




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