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

Abstract

Taq DNA polymerase has been used in PCR-based pathogen detection kits and the demand increased during the COVID-19 epidemic. In this study, it was 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 and ammonium sulfate precipitation and dialysis. Protein expression was analyzed by SDS-polyacrylamide gel electrophoresis. Enzyme activity was tested by PCR and qPCR in different buffers. Recombinant Taq DNA polymerase was purified, and purified protein and commercial Taq DNA polymerase were compared by SDS-PAGE. The PCR activity of the recombinant enzyme was shown in different commercial and in-house buffers. Enzyme was stable for at least 12 months and kept at -20ºC. Recombinant enzyme and commercial enzyme had PCR activity at dilutions up to 1/32. Milk powder, which is an economical alternative for IPTG, was tested and, use of milk powder equivalent to 3 mM lactose was as efficient as IPTG for the induction of the recombinant protein. Recombinant Taq DNA polymerase was tested in two DNA-based qPCR kits that were developed in our laboratory and was shown to perform as well as commercial enzymes. Finally, our enzyme was tested in our commercial SARS-CoV-2 diagnosis RT-qPCR kit using in-house produced primer-probe oligonucleotides and was shown to have equivalent efficacy to the commercial enzyme.