Erciyes Üniversitesi Fen Bilimleri Enstitüsü Fen Bilimleri Dergisi 1012-2354 Erciyes Üniversitesi 10.65520/erciyesfen.1757982 Gene Expression Genome Structure and Regulation Genetics (Other) Gen İfadesi Genom Yapısı ve Düzenlemesi Genetik (Diğer) Cloning and Protein Production of the Antigenic FliC Gene of Salmonella enterica serovar typhimurium ATCC 14028 Strain Salmonella enterica serovar typhimurium ATCC 14028 Suşuna ait Antijenik FliC Geninin Klonlanması ve Protein Üretimi https://orcid.org/0000-0003-0924-5365 Kılıç Selda ATATURK UNIVERSITY https://orcid.org/0000-0002-9720-1292 Arı Betül ATATURK UNIVERSITY https://orcid.org/0000-0001-8908-7293 Erdoğan Orhan ATATURK UNIVERSITY 03 13 2026 42 1 08 04 2025 03 09 2026 Copyright © 1985, Erciyes Üniversitesi Fen Bilimleri Enstitüsü Fen Bilimleri Dergisi 1985 Erciyes Üniversitesi Fen Bilimleri Enstitüsü Fen Bilimleri Dergisi

Proteins are large and complex biomolecules that perform a wide variety of biological functions in living organisms. These biomolecules play a critical role in many fields, including medicine, industry, food, the environment, and scientific research. Producing these proteins in their natural environment is both inefficient and extremely difficult. Using a molecular technique called recombinant DNA technology, proteins can be easily produced in appropriate quantities in an organism other than their natural source. The primary objective of this study was to clone the FliC gene from Salmonella enterica serovar typhimurium ATCC 14028 into the pET-SUMO vector for the first time and produce recombinant protein. For this purpose, the FliC gene obtained from S. typhimurium ATCC 14028 was inserted into the pET-SUMO vector, and recombinant protein production was carried out in Escherichia coli (E. coli) BL21(DE3) cells. The results showed that the culture induced with 1 mM IPTG provided the highest protein yield. The produced protein, approximately 70 kDa in size, was confirmed and purified using SDS-PAGE and nickel (Ni+2) affinity chromatography. In this study, the S. typhimurium FliC gene was successfully transferred to the pET-SUMO vector using the A-T cloning method for the first time, enabling the production of the targeted recombinant FliC protein.

Proteinler, canlı organizmalarda çok çeşitli biyolojik işlevleri yerine getiren büyük ve karmaşık biyomoleküllerdir. Bu biyomoleküller tıp, endüstri, gıda, çevre ve bilimsel araştırma gibi birçok alanda kritik bir rol oynar. Bu proteinleri doğal ortamlarında üretmek hem verimsiz hem de son derece zordur. Rekombinant DNA teknolojisi adı verilen bir moleküler teknik kullanılarak, proteinler doğal kaynakları dışındaki bir organizmada uygun miktarlarda kolayca üretilebilir. Bu çalışmanın temel amacı, Salmonella enterica serovar typhimurium ATCC 14028'in FliC genini ilk kez pET-SUMO vektörüne aktarıldı ve rekombinant protein üretmektir. Bu amaçla, S. typhimurium ATCC 14028'den alınan FliC geni pET-SUMO vektörüne klonlandı ve rekombinant protein üretimi Escherichia coli (E. coli) BL21(DE3) hücrelerinde gerçekleştirildi. Sonuçlar, 1 mM IPTG ile indüklenen kültürün en yüksek protein verimini sağladığını gösterdi. Üretilen protein yaklaşık 70 kDa olup, SDS PAGE ve nikel (Ni+2) afinite kromatografisi ile doğrulanmış ve saflaştırılmıştır. Bu araştırmada, S. typhimurium FliC geninin A-T klonlama yöntemi ile ilk kez pET-SUMO vektörüne aktarılarak hedeflenen rekombinant FliC proteinin üretimi başarıyla gerçekleştirilmiştir.

 Cloning FliC Purification Recombinant protein production Salmonella typhimurium FliC Klonlama Rekombinant protein üretimi Saflaştırma Salmonella typhimurium This study is supported by Atatürk University Scientific Research Projects (BAP) Unit 2022-10628 Mattick, J. S. (2003). Challenging the dogma: The hidden layer of non-protein-coding RNAs in complex organisms. BioEssays, 25(10), 930-939. https://doi.org/10.1002/bies.10332 Khan, S., Ullah, M. W., Siddique, R., Nabi, G., Manan, S., Yousaf, M., & Hou, H. (2016). Role of Recombinant DNA Technology to Improve Life. International Journal of Genomics, 2016, 1-14. https://doi.org/10.1155/2016/2405954 Unver, Y., Ari, B., Acar, M. et al. A self-inducible heterologous protein expression system in Komagataella phaffii (Pichia pastoris). 3 Biotech 14, 193 (2024). https://doi.org/10.1007/s13205-024-04039-x Eastwood, T. A., Baker, K., Streather, B. R., Allen, N., Wang, L., Botchway, S. W., Brown, I. R., Hiscock, J. R., Lennon, C., & Mulvihill, D. P. (2023). High-yield vesicle-packaged recombinant protein production from E. coli. Cell Reports Methods, 3(2), 100396. https://doi.org/10.1016/j.crmeth.2023.100396 McElwain, L., Phair, K., Kealey, C., & Brady, D. (2022). Current trends in biopharmaceuticals production in Escherichia coli. Biotechnology Letters, 44(8), 917-931. https://doi.org/10.1007/s10529-022-03276-5 Waugh, D. S. (2005). Making the most of affinity tags. Trends in Biotechnology, 23(6), 316-320. https://doi.org/10.1016/j.tibtech.2005.03.012 Li, M., Su, Z.-G., & Janson, J.-C. (2004). In vitro protein refolding by chromatographic procedures. Protein Expression and Purification, 33(1), 1-10. https://doi.org/10.1016/j.pep.2003.08.023 Chiu, T.-W., Peng, C.-J., Chen, M.-C., Hsu, M.-H., Liang, Y.-H., Chiu, C.-H., Fang, J.-M., & Lee, Y. C. (2020). Constructing conjugate vaccine against Salmonella Typhimurium using lipid-A free lipopolysaccharide. Journal of Biomedical Science, 27(1), 89. https://doi.org/10.1186/s12929-020-00681-8 Reeves, M. W., Evins, G. M., Heiba, A. A., Plikaytis, B. D., & Farmer, J. J. (1989). Clonal nature of Salmonella typhi and its genetic relatedness to other salmonellae as shown by multilocus enzyme electrophoresis, and proposal of Salmonella bongori comb. Nov. Journal of Clinical Microbiology, 27(2), 313-320. https://doi.org/10.1128/jcm.27.2.313-320.1989 Takaya, A., Yamamoto, T., & Tokoyoda, K. (2020). Humoral Immunity vs. Salmonella. Frontiers in Immunology, 10, 3155. https://doi.org/10.3389/fimmu.2019.03155 Simpson, K. M. J., Hill-Cawthorne, G. A., Ward, M. P., & Mor, S. M. (2018). Diversity of Salmonella serotypes from humans, food, domestic animals and wildlife in New South Wales, Australia. BMC Infectious Diseases, 18(1), 623. https://doi.org/10.1186/s12879-018-3563-1 Hohmann, E. L. (2001). Nontyphoidal Salmonellosis. Clinical Infectious Diseases, 32, 263-269 Jajere, S. M. (2019). A review of Salmonella enterica with particular focus on the pathogenicity and virulence factors, host specificity and antimicrobial resistance including multidrug resistance. Veterinary World, 12(4), 504-521. https://doi.org/10.14202/vetworld.2019.504-521 Ikeda, J. S., Schmitt, C. K., Darnell, S. C., Watson, P. R., Bispham, J., Wallis, T. S., Weinstein, D. L., Metcalf, E. S., Adams, P., O’Connor, C. D., & O’Brien, A. D. (2001). Flagellar Phase Variation of Salmonella enterica Serovar Typhimurium Contributes to Virulence in the Murine Typhoid Infection Model but Does Not Influence Salmonella -Induced Enteropathogenesis. Infection and Immunity, 69(5), 3021-3030. https://doi.org/10.1128/IAI.69.5.3021-3030.2001 Zarkani, A. A., López-Pagán, N., Grimm, M., Sánchez-Romero, M. A., Ruiz-Albert, J., Beuzón, C. R., & Schikora, A. (2020). Salmonella Heterogeneously Expresses Flagellin during Colonization of Plants. Microorganisms, 8(6), 815. https://doi.org/10.3390/microorganisms8060815 Honko, A. N., Sriranganathan, N., Lees, C. J., & Mizel, S. B. (2006). Flagellin Is an Effective Adjuvant for Immunization against Lethal Respiratory Challenge with Yersinia pestis. Infection and Immunity, 74(2), 1113-1120. https://doi.org/10.1128/IAI.74.2.1113-1120.2006 Butt, T. R., Edavettal, S. C., Hall, J. P., & Mattern, M. R. (2005). SUMO fusion technology for difficult-to-express proteins. Protein Expression and Purification, 43(1), 1-9. https://doi.org/10.1016/j.pep.2005.03.016 Ceylan, H. (2016). İnsan beyin asetilkolinesteraz enziminin klonlanması, e. coli’de ekspresyonu ve enzimin karakterizasyonu. Lai, J. Y., Klatt, S., & Lim, T. S. (2019). Potential application of Leishmania tarentolae as an alternative platform for antibody expression. Critical Reviews in Biotechnology, 39(3), 380-394. https://doi.org/10.1080/07388551.2019.1566206 García-Ortega, X., Cámara, E., Ferrer, P., Albiol, J., Montesinos-Seguí, J. L., & Valero, F. (2019). Rational development of bioprocess engineering strategies for recombinant protein production in Pichia pastoris (Komagataella phaffii) using the methanol-free GAP promoter. Where do we stand? New Biotechnology, 53, 24-34. https://doi.org/10.1016/j.nbt.2019.06.002 Huang, C.-J., Lin, H., & Yang, X. (2012). Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements. Journal of Industrial Microbiology and Biotechnology, 39(3), 383-399. https://doi.org/10.1007/s10295-011-1082-9 Chen, R. (2012). Bacterial expression systems for recombinant protein production: E. coli and beyond. Biotechnology Advances, 30(5), 1102-1107. https://doi.org/10.1016/j.biotechadv.2011.09.013 Achinas, S., Charalampogiannis, N., & Euverink, G. J. W. (2019). A Brief Recap of Microbial Adhesion and Biofilms. Applied Sciences, 9(14), 2801. https://doi.org/10.3390/app9142801 Wang, F., Deng, L., Huang, F., Wang, Z., Lu, Q., & Xu, C. (2020). Flagellar Motility Is Critical for Salmonella enterica Serovar Typhimurium Biofilm Development. Frontiers in Microbiology, 11, 1695. https://doi.org/10.3389/fmicb.2020.01695 Chilcott, G. S., & Hughes, K. T. (2000). Coupling of Flagellar Gene Expression to Flagellar Assembly in Salmonella enterica Serovar Typhimurium and Escherichia coli. Microbiology and Molecular Biology Reviews, 64(4), 694-708. https://doi.org/10.1128/MMBR.64.4.694-708.2000 Munir, A., Ahmed, N., Akram, M., Fujimura, N. A., Tahir, S., & Malik, K. (2023). Enhanced soluble expression of active recombinant human interleukin-29 using champion pET SUMO system. Biotechnology Letters, 45(8), 1001-1011. https://doi.org/10.1007/s10529-023-03402-x Taherkhani, R., Farshadpour, F., Makvandi, M., & Samarbafzadeh, A. R. (2014). Cloning of fliC Gene From Salmonella typhimurium in the Expression Vector pVAX1 and Evaluation of its Expression in Eukaryotic Cells. Jundishapur journal of microbiology, 7(11), e12351. https://doi.org/10.5812/jjm.12351 Zhao, T., Huang, H., Tan, P., Li, Y., Xuan, X., Li, F., … Yang, D. (2021). Enhancement of Solubility, Purification, and Inclusion Body Refolding of Active Human Mitochondrial Aldehyde Dehydrogenase 2. ACS Omega, 6(18), 12004–12013. https://doi.org/10.1021/acsomega.1c00577 Qu, J., Zhang, J., Zellmer, L., He, Y., Liu, S., Wang, C., Yuan, C., xu, N., Huang, H., & Liao, D. J. (2020). About three‐fourths of mouse proteins unexpectedly appear at a low position of SDS‐PAGE, often as additional isoforms, questioning whether all protein isoforms have been eliminated in gene‐knockout cells or organisms. Protein Science, 29(4), 964-976. https://doi.org/10.1002/pro.3823 Chen, J., Ng, S. M., Chang, C., Zhang, Z., Bourdon, J.-C., Lane, D. P., & Peng, J. (2009). P53 isoform Δ113p53 is a p53 target gene that antagonizes p53 apoptotic activity via BclxL activation in zebrafish. Genes & Development, 23(3), 278-290. https://doi.org/10.1101/gad.1761609 Komoriya, K., Shibano, N., Higano, T., Azuma, N., Yamaguchi, S., & Aizawa, S. (1999). Flagellar proteins and type III‐exported virulence factors are the predominant proteins secreted into the culture media of Salmonella typhimurium. Molecular Microbiology, 34(4), 767–779. https://doi.org/10.1046/j.1365-2958.1999.01639.x Mani, T., Joshi, J. B., Priyadharshini, R., Sharmila, J. S., & Uthandi, S. (2023). Flagellin, a plant-defense-activating protein identified from Xanthomonas axonopodis pv. Dieffenbachiae invokes defense response in tobacco. BMC Microbiology, 23(1), 284. https://doi.org/10.1186/s12866-023-03028-z Roumagnac, P., Weill, F.-X., Dolecek, C., Baker, S., Brisse, S., Chinh, N. T., … Achtman, M. (2006). Evolutionary History of Salmonella Typhi. Science, 314(5803), 1301–1304. https://doi.org/10.1126/science.1134933 Holt, K. E., Parkhill, J., Mazzoni, C. J., Roumagnac, P., Weill, F.-X., Goodhead, I., … Dougan, G. (2008). High-throughput sequencing provides insights into genome variation and evolution in Salmonella Typhi. Nature Genetics, 40(8), 987–993. https://doi.org/10.1038/ng.195