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.

Cost-effective, high-yield production of DNA polymerase for PCR application.

Polymerase Chain Reaction (PCR) is widely used for cloning, genetic engineering, mutagenesis, detection and diagnosis. A thermostable DNA polymerase is required for PCR. Here we describe low-cost and high-recovery production of DNA polymerase (-Pol).

Family B DNA polymerase from a hyperthermophilic archaeon Pyrobaculum calidifontis: cloning, characterization and PCR application.

The 2352 bp gene coding for 783 amino acid family B DNA polymerase from Pyrobaculum calidifontis was cloned and expressed in Escherichia coli. Expression of the gene resulted in the production of Pca-Pol in soluble fraction. After heat denaturation of the host proteins, the Pca-Pol was further purified by ion exchange and hydrophobic interaction chromatographies.