A Novel and Cost-Friendly Expression Method of Taq DNA Polymerase Using Milk Powder and Effective Utilization of The Enzyme in Diagnostic Kits

Authors : Suleyman Hekim, Arife Kaçıran, Ayşe Nur Akmehmet, Çağrı Şakalar, Sabriye Çanakçı, Ali Osman Beldüz

Pages : 193-214

Doi:10.35206/jan.1736420

View : 164 | Download : 181

Publication Date : 2025-12-31

Article Type : Research Paper

Abstract :Taq DNA polymerase has been used in PCR-based pathogen detection kits and the demand increased during the COVID-19 epidemic. This study aimed to produce recombinant Taq DNA polymerase in a low-cost and optimized setting and test its activity in various qPCR kits. Taq DNA polymerase gene was amplified by PCR and cloned into expression vectors. Recombinant protein expression was induced using IPTG, and purification was performed using heat incubation, ammonium sulfate precipitation, and dialysis. Purified Recombinant Taq DNA polymerase and commercial Taq DNA polymerase were compared. The PCR activity of the recombinant enzyme was shown in different commercial and in-house buffers. The enzyme was stable for at least 12 months and kept at -20 ºC. Milk powder, which is an economical alternative for IPTG, was tested. Using milk powder equivalent to 3 mM lactose was as efficient as IPTG to induce the recombinant protein. Recombinant Taq DNA polymerase was tested in two DNA-based qPCR kits developed in our laboratory and shown to perform as well as commercial enzymes. Finally, our enzyme was tested in our commercial SARS-CoV-2 diagnosis RT-qPCR kit and was shown to have equivalent efficacy as the commercial enzyme.
Keywords : Taq DNA Polimeraz, Rekombinant Enzim, PCR, qPCR Kiti, Süt Tozu, Ekspresyon İndüksiyonu

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