Engineering a DNA polymerase from Pyrobaculum calidifontis for improved activity, processivity and extension rate.

Positively charged amino acids in the DNA polymerase domain are important for interaction with DNA. Two potential residues in the palm domain of Pca-Pol, a DNA polymerase from Pyrobaculum calidifontis, were identified and mutated to arginine in order to improve the properties of this enzyme. The mutant proteins were heterologously produced in Escherichia coli. Biochemical characterization revealed that there was no significant difference in pH, metal ion, buffer preferences, 3' - 5' exonuclease activity and error rate of the wild-type and the mutant enzymes. However, the specific activity, processivity and extension rate of the mutant enzymes increased significantly. Specific activity of one of the mutants (G522R-E555R) was nearly 9-fold higher than that of the wild-type enzyme. These properties make G522R-E555R mutant enzyme a potential candidate for commercial applications.