A simple method for in-house Pfu DNA polymerase purification for high-fidelity PCR amplification

DNA polymerase is an enzyme that exhibits the lowest error rate in the 3' to 5' exonuclease (proofreading) activity during DNA synthesis in Polymerase Chain Reactions (PCRs). This study was aimed to express and purify DNA polymerase in a bacterial expression system under a simple purification method. polymerase gene sequence, derived from genomic DNA, was cloned and overexpressed in BL21 (DE3) pLysS. Upon overexpression, bacterial lysate containing the DNA polymerase was heated at 94°C for 5 minutes. DNA polymerase having high thermal stability was retained while the other bacterial proteins were denatured. The resulting thermo stable DNA polymerase was separated from the other debris of the denatured proteins by simple centrifugation. The enzymatic activity of the resulting DNA polymerase was estimated by comparing with the commercial DNA Polymerases. An estimated 50000 units of functional DNA polymerase was produced from a 400 ml culture. The in-house produced DNA Polymerase could be used for routine amplification that requires high-fidelity such as cloning and DNA sequencing.