Effect of C-Terminus Modification in Salmonella typhimurium FliC on Protein Purification Efficacy and Bioactivity
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Recombinant flagellin (FliC) has shown low efficacy in purification because of inclusion bodies formation and aggregation. We hypothesized preserving TLR5 binding site of FliC and removing some amino acids could be responsible for aggregation and solubility improvement. Hence, a bioinformatics study was performed to find hotspots in aggregate formation. Protein modeling was carried out by SWISS-MODEL and I-TASSER servers and models were compared by MATRAS server and Chimera 1.11.2. Gene modification was carried out based on bioinformatics studies. Genes, (truncated modified fliC (tmFliC) and full-length fliC (flFliC)), were cloned and expressed in pET-21a vector. Protein purification was carried out using HIS-Tag method. Proliferation assay and also induction of IL-8 in HEK293 cells were performed to confirm bioactivity function of tmFliC. Bioinformatics results showed that partial deletion of C-terminus may increase solubility without unfavorable effect on TLR5 recognition. Also, model comparison showed that this protein may preserve 3D structure. In addition, GlobPlot server demonstrated that tmFliC formed its globular domains which were important in TLR5 recognition. As we expected, high purification efficacy for tmFliC compared with flFliC was also obtained in experimental studies and a proper function for tmFliC was observed. The tmFliC enhanced cell proliferation in HEK293 cells compare with control after 24 h. Also, IL-8 level was increased with stimulation by tmFliC after 24 h. In conclusion, reducing hydrophobicity in C-terminus and deleting necessary amino acids for filament formation may increase protein solubility.
KeywordsFlagellin Protein modeling Purification efficacy Proliferation Bioinformatics
The authors would like to show their gratitude to Dr. Majid Esmaelizad from Razi Vaccine and Serum Research Institute.
Compliance with Ethical Standards
Conflict of interest
The authors confirm that they have no conflicts of interest.
- 4.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.Google Scholar
- 5.Faezi, S., Bahrmand, A. R., Mahdavi, M., Siadat, S. D., Nikokar, I., Sardari, S., & Holder, I. A. (2016). High yield overexpression, refolding, purification and characterization of Pseudomonas aeruginosa type B-flagellin: An improved method without sonication. International Journal of Molecular and Cellular Medicine, 5(1), 37.Google Scholar
- 12.Shander, A., Gromiha, M., Fawareh, H., & Sarai, A. (2004). ASA view: Solvent accessibility graphics for proteins. Bioinformatics, 51, 51.Google Scholar
- 15.Biasini, M., Bienert, S., Waterhouse, A., Arnold, K., Studer, G., Schmidt, T., Kiefer, F., Cassarino, T. G., Bertoni, M., Bordoli, L., & Schwede, T. (2014). SWISS-MODEL: Modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Research, 42, 252–258.CrossRefGoogle Scholar
- 22.Ahmadian, G., Ghahroudi, M. A., Rastgoo, N., & Mianji, G. R. (2005). Bacterial expression and functional characterization of a naturally occurring exon 6-less preprochymosin cDNA. Iranian Journal of Biotechnology, 3(1), 16–25.Google Scholar
- 23.Ikai, A. (1980). Thermostability and aliphatic index of globular proteins. The Journal of Biochemistry, 88(6), 1895–1898.Google Scholar
- 26.Smith, K. D., Andersen-Nissen, E., Hayashi, F., Strobe, K., Bergman, M. A., Barrett, S. L. R., Cookson, B. T., & Aderem, A. (2003). Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility. Nature Immunology, 4, 1247.CrossRefGoogle Scholar
- 29.Rigi, G., Beyranvand, P., Ghaedmohammadi, S., Heidarpanah, S., Akbari Noghabi, K., & Ahmadian, G. (2015) Comparison of the extracellular full-length and truncated recombinant protein A production in Escherichia coli BL21 (DE3). Journal of Paramedical Sciences, 6(3), 2008–4978.Google Scholar