A Novel and Cost-Friendly Expression Method of Taq DNA Polymerase Using Milk Powder and Effective Utilization of The Enzyme in Diagnostic Kits
Öz
Taq DNA polymerase has been used in PCR-based pathogen detection kits and the demand increased during the COVID-19 epidemic. This study 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, ammonium sulfate precipitation, and dialysis. Purified Recombinant Taq DNA polymerase and commercial Taq DNA polymerase were compared. The PCR activity of the recombinant enzyme was shown in different commercial and in-house buffers. The enzyme was stable for at least 12 months and kept at -20 ºC. Milk powder, which is an economical alternative for IPTG, was tested. Using milk powder equivalent to 3 mM lactose was as efficient as IPTG to induce the recombinant protein. Recombinant Taq DNA polymerase was tested in two DNA-based qPCR kits developed in our laboratory and shown to perform as well as commercial enzymes. Finally, our enzyme was tested in our commercial SARS-CoV-2 diagnosis RT-qPCR kit and was shown to have equivalent efficacy as the commercial enzyme.
Anahtar Kelimeler
Taq DNA Polymerase, Recombinant Enzyme, PCR, qPCR Kit, Milk Powder, Expression Induction
Destekleyen Kurum
This work was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) under TEYDEB 1501 Grant No: 3211159.
Etik Beyan
The authors have no financial conflicts of interest to declare.
Teşekkür
We are very grateful to Prof. Dr. ALİ OSMAN KILIÇ for providing the pUC18-Taq plasmid.
Kaynakça
- Chien, A., Edgar, D. B., & Trela, J. M. (1976). Deoxyribonucleic Acid Polymerase from the Extreme Thermophile Thermus aquaticus. 127(3), 1550–1557.
- Dabrowski, S. S., & Kur, J. (1998). Cloning and expression in Escherichia coli of the recombinant His-tagged DNA polymerases from Pyrococcus furiosus and Pyrococcus woesei. Protein Expression and Purification, 14(1), 131–138. https://doi.org/10.1006/prep.1998.0945
- DNA Polymerase Market Size To Hit Around USD 582.6 Mn By 2032. (n.d.). Retrieved March 22, 2024, from https://www.precedenceresearch.com/dna-polymerase-market
- Engelke, D. R., Krikos, A., Bruck, M. E., & Ginsburg, D. (1990). Purification of Thermus aquaticus DNA polymerase expressed in Escherichia coli. Analytical Biochemistry, 191(2), 396–400. https://doi.org/10.1016/0003-2697(90)90238-5
- Fang, N., Zhong, N., Yang, Y., Guo, Y., & Ji, S. (2016). Data of expression and purification of recombinant Taq DNA polymerase. https://doi.org/10.1016/j.dib.2016.08.032
- German Lopez. (2020, April 29). Why US coronavirus testing barely improved in April - Vox. https://www.vox.com/2020/5/1/21242589/coronavirus-testing-swab-reagent-supply-shortage
- Greicius, A., Baliutavicius, T., Lastauskiene, E., & Gudiukaite, R. (2022). Application of Milk Permeate as an Inducer for the Production of Microbial Recombinant Lipolytic Enzymes. Fermentation 2023, Vol. 9, Page 27, 9(1), 27. https://doi.org/10.3390/FERMENTATION9010027
- Innis, M. A., Myambo, K. B., Gelfand, D. H., & Brow, M. A. D. (1988). DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proceedings of the National Academy of Sciences of the United States of America, 85(24), 9436–9440. https://doi.org/10.1073/PNAS.85.24.9436
- Ishino, S., & Ishino, Y. (2014). DNA polymerases as useful reagents for biotechnology - The history of developmental research in the field. Frontiers in Microbiology, 5(AUG), 108662. https://doi.org/10.3389/FMICB.2014.00465/BIBTEX
- Khani, M. H., & Bagheri, M. (2020). Skimmed milk as an alternative for IPTG in induction of recombinant protein expression. Protein Expression and Purification, 170, 105593. https://doi.org/10.1016/J.PEP.2020.105593