A simple and efficient method for Taq DNA polymerase purification based on heat denaturation and affinity chromatography

Abstract

Taq DNA polymerase from Thermus aquaticus(Taq) is a widely used enzyme in molecular biology research and clinical diagnostics because of its utility in DNA amplification and sequencing assays. For laboratory large-scale production of enzymes, effort, time and cost are crucial factors. Here we describe a simple and efficient method for the production of high-quality recombinant Taq DNA polymerase using a combined method based on heat denaturation and nickel affinity chromatography. His-tagged Taq DNA polymerase was overexpressed in Escherichia coli and isolated in a first step by heat denaturation of the bacterial lysate, since Taq DNA polymerase is a thermo-resistant protein, it remains in the soluble fraction with a few bacterial protein contaminants. The isolated His-tagged Taq DNA polymerase was further purified by nickel-NTA chromatography leading to a high quality and purity enzyme with a comparable activity to most of commercially available counterparts. DNA-contaminated Taq DNA polymerases constitute a major concern especially during DNA amplification because they can lead to generation of non-specific products. Since Taq DNA polymerase is a DNA binding protein, it carries during purification process bacterial DNA contaminants. We treated the purified Taq DNA polymerase with DNase to end up with a high-quality DNA-free Taq DNA polymerase.