Cloning of the Ssap-NtrB prokaryotic gene into the eukaryotic expression vector pcDNA3.1 / V5 / His B vector

Year 2020, Volume: 22 Issue: 2, 547 - 554, 10.04.2020

Nelin Hacıoğlu Feray Köçkar

https://doi.org/10.25092/baunfbed.718256

Abstract

Suicide gene therapy has recently emerged as a method used in cancer treatments. These therapies utilized enzymes that are expressed in the cell. In this study, Staphylococcus saprophyticus supsp. saprophyticus Nitroreductase gene (Ssap-NtrB) was subcloned into the eukaryotic expression vector namely pcDNA3.1 / V5 / His B. For this purpose, Nitroreductase gene region was firstly amplified from the pET14B vector using PCR strategy and cloned into the pGEM-T-Easy vector. After this step, the Ssap-NtrB gene was restricted with KpnI/ApaI and was ligated into pcDNA3.1 / V5 / His B vector. Recombinant colonies were verified using KpnI/ApaI restriction enzymes. As a result, the Ssap-NtrB gene was cloned into pcDNA3.1/V5/His B vector and was readyfor use in suicide gene therapy in eukaryotic human cancer cells.

Keywords

Ssap-NtrB, Cloning, Nitroreductase, Expression Vector, pcDNA3.1/HisB

Supporting Institution

TUBITAK and partially Balikesir University

Project Number

110T754,113Z706 and 2017–024

Thanks

The study was supported by the Scientific and Technological Research Council of Turkey-TUBITAK (Grant No. 110T754 and 113Z706) and partially Balikesir University Research Project (2017–024).

Ssap-NtrB prokaryotik geninin ökaryotik ekspresyon vektörü pcDNA3.1 / V5 / His B içine klonlanması

Abstract

İntihar gen terapisi son zamanlarda kanser tedavilerinde kullanılan bir yöntem olarak karşımıza çıkmaktadır Bu terapilerde hücrede ifade olan enzimlerden faydalanılmaktadır. Bu çalışma kapsamında, Staphylococcus saprophyticus supsp. saprophyticus’tan elde edilen Ssap-NtrB geninin ökaryotik ekspresyon vektörü olan pCDNA3.1 / V5 / His B’ye klonlanması gerçekleştirilmiştir. Bu amaçla, ilk olarak genin içinde bulunduğu pET14B vektöründen restriksiyon kesimleri ile gen bölgesi alınarak, pGEM-T-Easy vektörüne aktarılmıştır. Bu adımdan sonra seçilen uygun enzimler olan KpnI/ApaI ile kesim yapılarak hazırlanmış olan pCDNA3.1 / V5 / His B’ye ligasyonu sağlanmıştır. Kontrol kesimleri ile gen klonlamanın doğrulanması sağlanmıştır. Sonuç olarak, intihar gen tedavisinde kullanıma hazır hale getirilen Ssap-NtrB geninin klonlanması ve kontrolü gerçekleştirilmiştir. Bu sayede ökaryotik kanser hücrelerinde kullanıma hazır hale getirilmiştir.

Keywords

Ssap-NtrB, nitroredüktaz, ekspresyon vektörü, pcDNA3.1/HisB

Project Number

110T754,113Z706 and 2017–024

References

• Race, P.R., Lovering AL, White SA, Grove JI, Searle PF, Wrighton CW ve Hyde EI., Structural and mechanistic studies of Escherichia coli nitroreductase with the antibiotic nitrofurazone reversed binding orientations in different redox states of the enzyme, Journal of Biological Chemistry, 280(14), 13256-13264, (2005).
• Race, P.R., Lovering AL, White SA, Grove JI, Searle PF, Wrighton CW ve Hyde EI., Kinetic and structural characterisation of Escherichia coli nitroreductase mutants showing improved efficacy for the prodrug substrate CB1954, Journal of molecular biology, 368(2), 481-492, (2007).
• Bryant, C.ve McElroy, W., Chemistry and biochemistry of flavoenzymes, CRC Press Boca Raton, FL, pp. 295-297,(1991).
• Bryant, D., McCalla, D., Leeksma, M. ve Laneuville, P., Type I nitroreductases of Escherichia coli, Canadian Journal of Microbiology, 27(1), 81-86, (1981).
• Goodwin, A., Kersulyte D, Sisson G, Veldhuyzen van Zanten SJ, Berg DE ve Hoffman PS, Metronidazole resistance in Helicobacter pylori is due to null mutations in a gene (rdxA) that encodes an oxygen‐insensitive NADPH nitroreductase, Molecular microbiology, 28(2), 383-393, (1998).
• Zenno, S., Koike H, Kumar AN, Jayaraman R, Tanokura M ve Saigo K., Biochemical characterization of NfsA, the Escherichia coli major nitroreductase exhibiting a high amino acid sequence homology to Frp, a Vibrio harveyi flavin oxidoreductase, Journal of bacteriology, 178(15), 4508-4514, (1996).
• Zenno, S., Kobori, T., Tanokura, M. ve SAIGO, K., "Purification and characterization of NfrA1, a Bacillus subtilis nitro/flavin reductase capable of interacting with the bacterial luciferase, Bioscience, biotechnology, and biochemistry, 62(10), 1978-1987, (1998).
• Zenno, S., Saigo, K., Kanoh, H. ve Inouye, S., Identification of the gene encoding the major NAD (P) H-flavin oxidoreductase of the bioluminescent bacterium Vibrio fischeri ATCC 7744, Journal of bacteriology, 176(12), 3536-3543, (1994).
• Anlezark, G.M., Vaughan T, Fashola-Stone E, Michael NP, Murdoch H, Sims MA, Stubbs S, Wigley S ve Minton NP., Bacillus amyloliquefaciens orthologue of Bacillus subtilis ywrO encodes a nitroreductase enzyme which activates the prodrug CB 1954, Microbiology, 148(1), 297-306, (2002).
• Çelik, A. ve Yetiş, G., An unusually cold active nitroreductase for prodrug activations, Bioorganic & medicinal chemistry, 20(11), 3540-3550, (2012).
• Güngör T., Yetiş G., Önder C.F., Tokay E., Tok T.T., Çelik A., Ay M., Köçkar F., Prodrugs for nitroreductase based cancer therapy-1: Metabolite profile, cell cytotoxicity and molecular modeling interactions of nitro benzamides with Ssap-NtrB, Medicinal Chemistry, 14, 495-507, (2018).
• Güngör T., Önder C.F., Tokay E., Gülhan G.Ü., Hacıoğlu N., Tok T.T., Çelik A., Köçkar F., Ay M., Prodrugs For Nitroreductase Based Cancer Therapy-2: Novel amide/Ntr combinations targeting PC3 cancer cells, European Journal of Medicinal Chemistry, 171, 383-400, (2019).
• Tokay E., Kockar F., Identification of intracellular pathways through which TGF-b1 upregulates URG-4/URGCP gene expression in hepatoma cells, Life Science, 144, 121-128, (2016).
• Tokay E., Kockar F., SP1 is a transcriptional regulator of URG-4/URGCP gene in hepatocytes, Mol Cell Biochem, 423(1-2):75-83, (2016).
• Alper M., Kockar F., IL-6 upregulates a disintegrin and metalloproteinase with thrombospondin motifs 2 (ADAMTS-2) in human osteosarcoma cells mediated by JNK pathway, Molecular Celular Biochemistry, 393, 165–175, (2014).