Production of Taq polymerase for use in real-time PCR

Background: Taq polymerase is a key enzyme for real-time PCR (qPCR). Given the high cost of commercial Taq polymerase, the development of its simple and effective production is an important matter. The reproducible laboratory production of these enzymes can reduce dependence on commercial products and advance further development of molecular diagnostic systems. Objective: The aim of this study was to optimize the process of recombinant Taq polymerase production using Escherichia coli culture with further evaluation of its functional activity and suitability for qPCR. Methods: Taq polymerase was expressed in E. coli BL21 using a pET-30a(+)–based plasmid with polyhistidine tags. The enzyme was purified by Ni-NTA affinity chromatography, followed by chromatographic purification. The purity of Taq polymerase was assessed by SDS-PAGE. The functional activity of the enzyme was evaluated by qPCR, using the human β-globin gene as the target sequence. Results: As a result of process optimization Taq polymerase with up to 90% purity was obtained at a concentration of 70.3 ± 3.7 mg/l. The enzyme enabled the specific amplification of an 80-bp β-globin fragment and demonstrated amplification efficiency of up to 97%. The enzyme retained its functional activity at room temperature (18–22°C) for two months. Conclusions: Optimized procedure ensured a reproducible manufacturing to obtain a functionally active and stable recombinant Taq polymerase. The enzyme amplification efficiency demonstrates its potential for use in qPCR and in the development of molecular diagnostic test systems. Keywords: biotechnology, E. coli BL21, human β-globin gene, real-time PCR, recombinant protein, Taq polymerase