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

Abstract

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 Pyrobaculum calidifontis DNA polymerase (Pca-Pol). The gene was cloned in pET-28a and expressed in Escherichia coli BL21CodonPlus. Gene expression conditions were optimized. Eventually, gene expression was induced with 0.1 mM IPTG for 3 hours at 37 °C. Recombinant Pca-Pol produced was purified to homogeneity by immobilized metal-ion affinity chromatography yielding around 9000 U of Pca-Pol per liter of the culture with a recovery of 92%. Stability and PCR amplification efficiency of Pca-Pol was tested under various storage conditions with highest efficiency in 25 mM Tris-Cl buffer (pH 8.5) containing 0.1% Tween 20, 0.2 mg/mL BSA and 20% glycerol. Under this condition, no loss in PCR activity of Pca-Pol was observed, even after one year of storage. Repeated freeze-thaw, however, deteriorated enzyme activity of Pca-Pol. 55% PCR amplification activity retained after 7 prolong freeze-thaw cycles (freezing overnight at −20 °C and thawing for 45 minutes at 28 °C). Purified Pca-Pol possessed 3′–5′ exonuclease (proofreading) activity and is expected to have greater fidelity as compared to Taq polymerase which does not have proofreading activity.

Keywords:

• DNA polymerase
• expression optimization
• polymerase chain reaction (PCR)
• purification
• Pyrobaculum calidifontis
• stability