A novel approach for high-level expression and purification of GST-fused highly thermostable Taq DNA polymerase in Escherichia coli

• Published in: Archives of microbiology 2020-08, Vol.202 (6), p.1449-1458
• Main Authors: Din, Rahman Ud, Khan, Muhammad Ismail, Jan, Asad, Khan, Shah Anwar, Ali, Irshad
• Format: Article
• Language: English
• Subjects: Automation; Base Sequence; Biochemistry; Biology; Biomedical and Life Sciences; Biotechnology; Cell Biology; Cloning, Molecular - methods; Copying; Deoxyribonucleic acid; Diagnostic systems; DNA; DNA polymerase; DNA, Bacterial - genetics; DNA-directed DNA polymerase; E coli; Ecology; Enzymes; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression - genetics; Life Sciences; Microbial Ecology; Microbiology; Molecular biology; Nucleic acids; Nucleotides; Original Paper; Peptides; Polymerase chain reaction; Polymerase Chain Reaction - methods; Polymers; Protein purification; Proteomics; Purification; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Ribonucleic acid; RNA; Taq Polymerase - chemistry; Taq Polymerase - genetics; Taq Polymerase - metabolism
• ISSN: 0302-8933; 1432-072X
• DOI: 10.1007/s00203-020-01860-9

Polymerases are enzymes that synthesize long chains or polymers of nucleic acids including DNA or RNA from nucleotides. They assemble nucleic acids by copying a DNA or RNA template strand using base-pairing interactions. One of the polymerase enzymes, Taq DNA polymerase, originally isolated from Thermus aquaticus ( Taq ) is a widely used enzyme in molecular biology so far. The thermostable properties of this enzyme have contributed majorly to the specificity, automation, and efficacy of the polymerase chain reaction (PCR), making it a powerful tool for today’s molecular biology researches across the globe. The purification of Taq DNA polymerase from the native host results in low yield, more labor and time consumption. Therefore, many studies have been previously conducted to obtain this enzyme using alternative hosts. So far, all the existing methodologies are more laborious, time-consuming and require heavy expense. We used a novel approach to purify the enzyme with relatively high efficiency, yield and minimum time consumption using Escherichia coli ( E. coli ) as an alternative host. We cloned a 2500 base pair Taq DNA polymerase gene into pGEX-4T-1 vector, containing a GST-tag, downstream of tac promoter and overexpressed it using isopropyl β-d-1-thiogalactopyranoside (IPTG) as an inducer. The enzyme was efficiently purified using novel chromatography approaches and was used in routine PCR assays in our laboratory. Our findings suggest a novel approach to facilitate the availability of polymerases for molecular and diagnostic studies. In the future, it may be used for the purification of other recombinant peptides or proteins used in structural biology and proteomics-based researches.