RESUMO
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.
Assuntos
Pyrobaculum , Pyrobaculum/genética , Análise Custo-Benefício , Reação em Cadeia da Polimerase/métodos , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Engenharia Genética , Escherichia coli/metabolismoRESUMO
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.
Assuntos
Pyrobaculum , Pyrobaculum/genética , DNA Polimerase Dirigida por DNA/química , Escherichia coli/genética , Escherichia coli/metabolismo , AminoácidosRESUMO
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. Activity gel analysis showed the presence of a catalytically active polypeptide of about 90 kDa. The mass of the protein, determined by Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry was found to be 89,156 Da. The isoelectric point of the enzyme was found to be 6.13. The optimal pH and magnesium ion concentration for the enzyme activity were 8.5 and 4mM, respectively. Unlike other commercially available DNA polymerases the enzyme activity of Pca-Pol was inhibited by monovalent cations such as ammonium and potassium. The half-life of the polymerase at 95 °C and 100 °C was 4.5h and 0.5h, respectively. The enzyme possessed 3'â5' exonuclease activity and was able to amplify, under suitable conditions, up to 7.5 kb DNA fragments by polymerase chain reaction which makes it a potential candidate for amplification of long DNA fragments.