Production and catalytic evaluation of a recombinant Taq DNA polymerase as an alternative for biotechnological applications
Keywords:
Taq Polymerase, Recombinant proteins, Gene Expression, Biotechnology, Polymerase Chain Reaction, Scientific and Technical AutonomyAbstract
Recombinant Taq DNA polymerase is a key tool in molecular biology. The COVID-19 pandemic highlighted the vulnerability associated with dependence on imported critical reagents. This study aimed to develop an optimized protocol for the heterologous expression, purification, and functional validation of recombinant Taq polymerase to support local production capacity. Escherichia coli BL21(DE3) cells were transformed with the pOpenTaq plasmid, and expression was induced under varying IPTG concentrations and incubation temperatures to determine optimal conditions. The purification protocol combined heat and ammonium sulfate precipitation followed by size-exclusion chromatography. Enzymatic activity was assessed via endpoint PCR using various DNA templates and compared to a commercial Taq polymerase. Maximum expression was achieved at 37 °C with 1 mM IPTG after 4 hours of induction. The purified enzyme reached >90% purity and exhibited comparable amplification efficiency to the commercial control, including with complex clinical samples. The developed method is efficient, reproducible, and amenable to scale-up, representing a viable approach for the local production of thermostable polymerases and other strategic molecular reagents.
Author Biographies
Luna Torres-Hichster, Universidad El Bosque
Laboratorio de Parasitología Molecular, Vicerrectoría de Investigaciones, Universidad El Bosque
Carlos Nieto-Clavijo, Universidad El Bosque
Laboratorio de Parasitología Molecular, Vicerrectoría de Investigaciones, Universidad El Bosque
Liliana Morales, Universidad El Bosque
Laboratorio de Parasitología Molecular, Vicerrectoría de Investigaciones, Universidad El Bosque
Jacqueline Chaparro-Olaya, Universidad El Bosque
Laboratorio de Parasitología Molecular, Vicerrectoría de Investigaciones, Universidad El Bosque
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