One-step rapid production of DNA polymerase from Pyrococcus furiosus in Escherichia coli system under optimized culture conditions.

Abstract

The Pyrococcus furiosus (Pfu) DNA polymerase is an enzyme widely used in PCR due to its high fidelity, thermal stability, and ability to amplify even minute amounts of DNA with exceptional specificity and sensitivity. This study addresses the growing demand for efficient and cost-effective production of Pfu polymerase by optimizing its recombinant expression in Escherichia coli BL21 star (DE3) using a synthetic gene in the pD451-SR plasmid. Key parameters, including IPTG concentration (0.4 mM), post-induction incubation time (12 h), and cell density prior to induction (OD₆₀₀ of 0.4), were systematically optimized to maximize yield and activity. A rapid and straightforward purification protocol was developed, utilizing a boiling-mediated lysis method that effectively purified Pfu polymerase within 20 minutes. A total of 795 mg/L of pure Pfu polymerase under optimized conditions, led to a 30-fold increase in protein yield with an enzyme activity of 2210 units, with SDS-PAGE and western blot analyses confirming a single protein band at 90 kDa. The optimized production and purification methods significantly enhance the time and cost-efficiency of Pfu polymerase production, making it highly suitable for industrial-scale applications. This streamlined approach positions Pfu polymerase as a valuable tool for routine PCR, cloning, mutagenesis, and advanced applications.