Recombinant Expression of Taq DNA Polymerase Using pBluescript SK(+) and Chromatography-free Purification: A Cost-effective Strategy for Low-resource Laboratories

Abstract

Taq DNA polymerase is a cornerstone of molecular biology. However, its production typically relies on proprietary vectors and affinity-based purification systems. These requirements limit access in educational and under-resourced environments. In this study, we demonstrate the first experimentally confirmed expression of the thermostable Taq DNA polymerase using the widely available, open-access pBluescript SK(+) vector. This vector, traditionally used for blue-white screening, was repurposed as a protein expression platform in E. coli. Expression was paired with a simplified purification strategy involving heat denaturation and ammonium sulfate precipitation. This chromatography-free strategy requires no affinity tags or vendor-specific reagents. It yielded enzymatically active Taq DNA polymerase with a mean purity of 90.1 ± 2.1%, as determined by SDS-PAGE densitometry. Functional validation through PCR amplification and thermostability assays confirmed that the enzyme is comparable in PCR performance to commercial Ex Taq (Takara Bio). We also assessed the workflow’s performance across different production volumes, consistency of results, and compatibility with basic laboratory infrastructure. Compared to commercial enzyme, our protocol reduced the cost-per-reaction by 70–85%, depending on the amount of enzyme used. These findings demonstrate that pBluescript SK(+), typically reserved for cloning, can support effective recombinant protein production when paired with optimized conditions. This study provides a low-cost, accessible solution for molecular biology instruction and research in under-resourced settings.