Purification Protocol
This single step purification scheme for Taq DNA polymerase is very efficient due to the thermal stability of the overexpressed enzyme relative to native E. coli proteins. Heat treatment of the cell lysate denatures all endogenous proteins, which are then easily removed by centrifugation. The efficiency of this process is clearly demonstrated by comparing the SDS-PAGE of the proteins present in the cleared lysate relative to those present in the crude cell lysate (Figure 1). This protein is essentially homogeneous as determined by Coomassie blue staining. More importantly, it exhibits high specific activity (Table 1) when assayed for thermostable DNA polymerase activity, and lacks any nicking or other DNAse activities (data not shown). The enzyme is found to be sensitive to freezing unless stored at subzero temperatures in 50% glycerol. Fluorometry and electrophoretic analysis of the enzyme sample showed the presence of the original plasmid DNA (pTAQ) in the enzyme preparation. To eliminate plasmid DNA, the enzyme was loaded onto a Pharmacia LKB FPLC Mono-Q column (Pharmacia LKB Biochrom Ltd., Cambridge, England) in buffer A (50 mM Tris-HCl, pH 8.0). Taq polymerase enzyme was eluted using a 0 to 0.5 M NaCl gradient. The enzyme eluted at 200 mM NaCl with no loss of protein. This highly purified enzyme is very stable and can be stored at -20 oC in 50% glycerol for six months or more.Enzyme Activity
Table 1 demonstrates the large amounts of enzymatic activity purified from a single 100 mL culture of cells. A yield of 8 million units of activity is typical using this procedure when activity is compared to commercially available enzyme by the titration assay described. Care must be taken when titrating enzymatic activity since the enzyme is present at fairly high concentrations. As seen in Figure 3, excessively high levels of enzyme can inhibit the DNA polymerase reaction. This gives the false indication that no polymerase activity is present unless the enzyme is diluted sufficiently to overcome this inhibition. Inhibition is especially noticeable when amplification products of larger size are used.
Cloning and expression of the Thermus aquaticus DNA polymerase gene in E. coli allows rapid, simple purification of large quantities of research quality enzyme in a single step. The procedure described provides a ready source of this essential tool for molecular research.