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Effect of C-Terminus Modification in Salmonella typhimurium FliC on Protein Purification Efficacy and Bioactivity

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Abstract

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.

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Acknowledgements

The authors would like to show their gratitude to Dr. Majid Esmaelizad from Razi Vaccine and Serum Research Institute.

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Authors and Affiliations

  1. Department of Genomics and Genetic Engineering, Razi Vaccine and Serum Research Institute (RVSRI), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

    Mohammad-hosein Khani, Masoumeh Bagheri & Azadeh Zahmatkesh

  2. Department of Biology, Nourdanesh Institute of Higher Education, Meymeh, Iran

    Ali Dehghanian

  3. Department of Microbiology, Razi Vaccine and Serum Research Institute (RVSRI), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

    Soheila Moradi Bidhendi

  4. Department of Central Laboratory, Razi Vaccine and Serum Research Institute (RVSRI), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

    Zahra Salehi Najafabadi

  5. Department of Immunology, Razi Vaccine and Serum Research Institute (RVSRI), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran

    Reza Banihashemi

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  1. Mohammad-hosein Khani
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Correspondence to Masoumeh Bagheri.

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Khani, Mh., Bagheri, M., Dehghanian, A. et al. Effect of C-Terminus Modification in Salmonella typhimurium FliC on Protein Purification Efficacy and Bioactivity. Mol Biotechnol 61, 12–19 (2019). https://doi.org/10.1007/s12033-018-0135-y

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  • DOI: https://doi.org/10.1007/s12033-018-0135-y

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