Vol.:(0123456789) 1 3 Molecular Biotechnology https://doi.org/10.1007/s12033-018-0135-y ORIGINAL PAPER Efect of C-Terminus Modifcation in Salmonella typhimurium FliC on Protein Purifcation Efcacy and Bioactivity Mohammad‑hosein Khani 1  · Masoumeh Bagheri 1  · Ali Dehghanian 2  · Azadeh Zahmatkesh 1  · Soheila Moradi Bidhendi 3  · Zahra Salehi Najafabadi 4  · Reza Banihashemi 5 © Springer Science+Business Media, LLC, part of Springer Nature 2018 Abstract Recombinant fagellin (FliC) has shown low efcacy in purifcation 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 fnd 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 modifcation was carried out based on bioinformatics studies. Genes, (truncated modifed fiC (tmFliC) and full-length fiC (fFliC)), were cloned and expressed in pET-21a vector. Protein purifcation was carried out using HIS-Tag method. Proliferation assay and also induction of IL-8 in HEK293 cells were performed to confrm bioactivity function of tmFliC. Bioinformatics results showed that partial deletion of C-terminus may increase solubility without unfavorable efect 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 purifcation efcacy for tmFliC compared with fFliC 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 flament formation may increase protein solubility. Keywords Flagellin · Protein modeling · Purifcation efcacy · Proliferation · Bioinformatics Introduction Flagellin, the molecular motor of fagellated bacteria, helps in movement, adhesion and invasion [1]. Flagellin has been considered as pathogen associated molecular pattern (PAMP) which can stimulate one pattern recognition recep- tor (PRR) in a vast array of eukaryotic cells and this PRR is toll like receptor 5 (TLR5) and has a crucial role in immune system. FliC as the main protein of Salmonella fagellin is encoded by a conserved gene which can be detected in many bacterial species. In a wide experimental research, Salmonella typhimurium flagellin has shown promising applications in vaccine products. FliC induces dimerization of TLR5 when FliC binds it. This dimerization will activate signal transduction of consequent number of proinfamma- tory cytokines such as TNF-α and IL-6 [2]. Despite similar- ity in protein sequence of diferent organism, these homolo- gous proteins of diferent species show diferent properties some of which are defcient in activating TLR5 [3]. * Masoumeh Bagheri m.bagheri@rvsri.ac.ir 1 Department of Genomics and Genetic Engineering, Razi Vaccine and Serum Research Institute (RVSRI), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran 2 Department of Biology, Nourdanesh Institute of Higher Education, Meymeh, Iran 3 Department of Microbiology, Razi Vaccine and Serum Research Institute (RVSRI), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran 4 Department of Central Laboratory, Razi Vaccine and Serum Research Institute (RVSRI), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran 5 Department of Immunology, Razi Vaccine and Serum Research Institute (RVSRI), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran