Taq DNA Polimeraz enziminin Süt Tozu Kullanılarak Yeni ve Maliyet Dostu Bir Yöntem ile ifade edilmesi ve Enzimin Tanı Kitlerinde Etkin Kullanımı

Year 2025, Volume: 8 Issue: 2, 193 - 214, 31.12.2025

Suleyman Hekim , Arife Kaçıran , Ayşe Nur Akmehmet , Çağrı Şakalar , Sabriye Çanakçı , Ali Osman Beldüz

https://doi.org/10.35206/jan.1736420

https://izlik.org/JA46FC72PL

Abstract

Taq DNA polimeraz, PCR tabanlı patojen tespit kitlerinde kullanılmış olup COVID-19 salgını sırasında talep artmıştır. Bu çalışma, düşük maliyetli ve optimize edilmiş bir ortamda rekombinant Taq DNA polimeraz üretmeyi ve aktivitesini çeşitli qPCR kitlerinde test etmeyi amaçlamıştır. Taq DNA polimeraz geni PCR ile çoğaltılmış ve ekspresyon vektörlerine klonlanmıştır. Rekombinant protein ekspresyonu IPTG kullanılarak indüklenmiş ve saflaştırma ısı inkübasyonu, amonyum sülfat çöktürmesi ve diyaliz kullanılarak gerçekleştirilmiştir. Saflaştırılmış rekombinant Taq DNA polimeraz ve ticari Taq DNA polimeraz karşılaştırılmıştır. Rekombinant enzimin PCR aktivitesi farklı ticari ve şirket içi tamponlarda gösterilmiştir. Enzim en az 12 ay stabil kalmış ve -20 ºC'de tutulmuştur. IPTG için ekonomik bir alternatif olan süt tozu test edilmiştir. 3 mM laktoza eşdeğer süt tozu kullanmak, rekombinant proteini indüklemek için IPTG kadar etkili olmuştur. Rekombinant Taq DNA polimerazı, laboratuvarımızda geliştirilen iki DNA tabanlı qPCR kitinde test edildi ve ticari enzimler kadar iyi performans gösterdiği gösterildi. Son olarak, enzimimiz ticari SARS-CoV-2 tanı RT-qPCR kitinde test edildi ve ticari enzimle eşdeğer etkinliğe sahip olduğu gösterildi.

Keywords

Taq DNA Polimeraz , Rekombinant Enzim , PCR , qPCR Kiti , Süt Tozu , Ekspresyon İndüksiyonu

A Novel and Cost-Friendly Expression Method of Taq DNA Polymerase Using Milk Powder and Effective Utilization of The Enzyme in Diagnostic Kits

https://izlik.org/JA46FC72PL

Abstract

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.

Keywords

Taq DNA Polymerase , Recombinant Enzyme , PCR , qPCR Kit , Milk Powder , Expression Induction

Ethical Statement

The authors have no financial conflicts of interest to declare.

Supporting Institution

This work was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) under TEYDEB 1501 Grant No: 3211159.

Thanks

We are very grateful to Prof. Dr. ALİ OSMAN KILIÇ for providing the pUC18-Taq plasmid.

References

• 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
• Lal, M., & Ranjan, S. (2020). ISOLATION OF Taq POLYMERASE GENE & CLONING INTO E.coli USING pGEMT VECTOR. World Journal of Pharmaceutical Research Www.Wjpr.Net, 9, 938. https://doi.org/10.20959/wjpr202010-18536
• Lawyer, F. C., Stoffel, S., Saiki, R. K., Chang, S.-Y., Landre, P. A., Abrarnson, R. D., & Gelfand, D. H. (1993). High-level Expression, Purification, and Thermus aquatlcus DNA Polymerase and a Exonuclease Activity. 2(4), 275–287.
• Martín-Alonso, S., Frutos-Beltrán, E., & Menéndez-Arias, L. (2021). Reverse Transcriptase: From Transcriptomics to Genome Editing. Trends in Biotechnology, 39(2), 194–210. https://doi.org/10.1016/j.tibtech.2020.06.008
• Mascuch, S. J., Fakhretaha-Aval, S., Bowman, J. C., Ma, M. T. H., Thomas, G., Bommarius, B., Ito, C., Zhao, L., Newnam, G. P., Matange, K. R., Thapa, H. R., Barlow, B., Donegan, R. K., Nguyen, N. A., Saccuzzo, E. G., Obianyor, C. T., Karunakaran, S. C., Pollet, P., Rothschild-Mancinelli, B., … Cameron, C. E. (2020). A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits. https://doi.org/10.1074/jbc.RA120.015434
• Moazen, F., Rastegari, A., Hoseini, S., Panjehpour, M., Miroliaei, M., & Sadeghi, H. M. (2012). Optimization of Taq DNA polymerase enzyme expression in Escherichia coli. Advanced Biomedical Research, 1(1), 82. https://doi.org/10.4103/2277-9175.103004
• Nayak, K. K., & Tiwari, A. (2012). Expression of Taq Polymerase I Gene in Escherichia coli BL21.
• Osman Elbek. (2020). COVID-19 Outbreak and Turkey. Turk Thorac J, 21(3), 215–221. https://doi.org/10.5152/TurkThoracJ.2020.20073
• Philip Ferralli, John Duick Egan, & Floyd Lester Erickson. (2007). Making Taq DNA polymerase in the undergraduate biology laboratory. Bios, 78, 69–74.
• Pluthero, F. G. (1993). Rapid purification of high-activity Taq DNA polymerase. Nucleic Acids Research, 21(20), 4850. https://doi.org/10.1093/NAR/21.20.4850
• Samman, N., Al-Muhalhil, K., & Nehdi, A. (2023). A simple and efficient method for Taq DNA polymerase purification based on heat denaturation and affinity chromatography. Journal of King Saud University - Science, 35(3). https://doi.org/10.1016/J.JKSUS.2023.102565
• Schrader, C., Schielke, A., Ellerbroek, L., & Johne, R. (2012). PCR inhibitors – occurrence, properties and removal. Journal of Applied Microbiology, 113(5), 1014–1026. https://doi.org/10.1111/J.1365-2672.2012.05384.X
• Stanton, J. A. L., O’Brien, R., Hall, R. J., Chernyavtseva, A., Ha, H. J., Jelley, L., Mace, P. D., Klenov, A., Treece, J. M., Fraser, J. D., Clow, F., Clarke, L., Su, Y., Kurup, H. M., Filichev, V. V., Rolleston, W., Law, L., Rendle, P. M., Harris, L. D., … Blaikie, R. (2022). Uncoupling Molecular Testing for SARS-CoV-2 From International Supply Chains. Frontiers in Public Health, 9. https://doi.org/10.3389/FPUBH.2021.808751
• W GrahamID, T. G., Dugast-DarzacqID, C., Dailey, G. M., NguyenlaID, X. H., Van DisID, E., EsbinID, M. N., Abidi, A., Stanley, S. A., Darzacq, X., & TjianID, R. (2021). Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection. https://doi.org/10.1371/journal.pone.0246647