Expression Strategy of Soluble Recombinant Human TGF-β3 in Escherichia coli: sfGFP -Fusion Tag

Year 2023, Volume: 27 Issue: 1, 204 - 213, 28.02.2023

Sema Bilgin

https://doi.org/10.16984/saufenbilder.1096298

Abstract

Transforming growth factor-beta 3 (TGF-β3) is an important cytokine involved in various biological processes. TGF-β3 is used as a scar-reducing antifibrotic agent for acute and chronic wounds and fibrosing disorders. TGF-β3, a valuable therapeutic protein, is produced recombinantly in different expression systems. TGF-β3, produced in the Escherichia coli (E. coli) expression system, widely used due to its various advantages in recombinant production, is commercially available. However, the main problem encountered in protein expression in E. coli cells is the formation of an inclusion body. Various approaches have been developed to solve this problem. The use of a fusion tag is one of the most powerful strategies used to obtain protein in the soluble active form in the E. coli expression system. Superfolder GFP (sfGFP) is one of the fusion tags used to increase the solubility of the fusion partner in E. coli. In this study, TGF-β3 with sfGFP fusion tag (sfGFP-TGFβ3) was successfully produced in soluble form in E. coli BL21 (DE3) in high yield and purity for the first time. Purified protein was identified by western blot and SDS-PAGE. 20 mg of protein with 98% purity was obtained from 1 L of bacterial culture. It was determined that the obtained high purity protein did not have a cytotoxic effect on BJ normal human skin fibroblast cells. The impact of sfGFP-TGFβ3 fusion protein on wound healing was evaluated with in vitro scratch wound healing assay. The results showed that the sfGFP-TGFβ3 fusion protein produced in soluble form in the E. coli expression system has the potential to support the wound healing process.

Keywords

Escherichia coli, fusion tag, inclusion body, sfGFP, TGF-β3, recombinant protein

References

• [1] M. K. Lichtman, M. Otero-Vinas, V. Falanga, “Transforming growth factor-beta (TGF-b) isoforms in wound healing and fibrosis”, Wound Repair and Regeneration, vol. 24, pp. 215–222, 2016.
• [2] M. F. Gisby, P. Mellors, P. Madesis, M. Ellin, H. Laverty, S. O’Kane, M. W. J. F. A. Day, “A synthetic gene increases TGFb3 accumulation by 75-fold in tobacco chloroplasts enabling rapid purification and folding into a biologically active molecule”, Plant Biotechnology Journal, vol. 9, pp. 618–628, 2011.
• [3] S. O’kane, M. W. J. Ferguson, “Transforming Growth Factor βs and Wound Healing”, International Journal of Biochemistry & Cell Biology, vol. 29, no. 1, pp. 63-78. 1997.
• [4] B. Choi, Y. Lee, J. Pi, Y. Jeong, K. Baek, J. Yoon, “Overproduction of recombinant human transforming growth factor beta 3 in Chinese hamster ovary cells”, Protein Expression and Purification, vol. 110, pp. 102–106, 2015.
• [5] N. L. Occleston, H. G. Laverty, S. O'Kane, M. W. J. Ferguson, “Prevention and reduction of scarring in the skin by Transforming Growth Factor beta 3 (TGFβ3): from laboratory discovery to clinical pharmaceutical”, Journal of Biomaterials Science Polymer Edition, vol. 19, no. 8, pp. 1047-1063, 2012.
• [6] J. Bush, K. So, T. Mason, N. L. Occleston, S. O’Kane, M. W. J. Ferguson, “Therapies with Emerging Evidence of Efficacy: Avotermin for the Improvement of Scarring”, Dermatology Research and Practice, vol. 2010, 2010.
• [7] A. Nauta, G. C. Gurtner, M. T Longaker, “The evolving role of avotermin in scar prevention”, Expert Review of Hematology, vol. 6, no. 2, pp. 149–152, 2011.
• [8] M. Zhou, W. Shi, F. Yu, Y. Zhang, B. Yu, J. Tang, Y. Yang, Y. Huang, Q. Xiang, Q. Zhang, Z. Yao, Z. Su, “Pilot-scale expression, purification, and bioactivity of recombinant human TGF-β3 from Escherichia coli”, European Journal of Pharmaceutical Sciences, vol. 127, pp. 225–232, 2019.
• [9] L. F. Vallejo, U. Rinas, “Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins”, Microbial Cell Factories, vol. 3, no.11, 2004.
• [10] V. Paraskevopoulou, F. H. Falcone, “Polyionic Tags as Enhancers of Protein Solubility in Recombinant Protein Expression”, Microorganisms, vol. 6, no. 47, 2018.
• [11] D. K. Yadav, N. Yadav, S. Yadav, S. Haque, N. Tuteja, ”An insight into fusion technology aiding efficient recombinant protein production for functional proteomics”, Archives of Biochemistry and Biophysics, vol. 612, pp. 57-77, 2016.
• [12] S. M. Singh, A. K. Panda, “Solubilization and Refolding of Bacterial Inclusion Body Proteins”, Journal of Bioscience and Bioengineering, vol. 99, no. 4, pp. 303–310, 2005.
• [13] P. Singhvi, A. Saneja, S. Srichandan, A. K. Panda, “Bacterial Inclusion Bodies: A Treasure Trove of Bioactive Proteins”, Trends in Biotechnology, vol. 38, no. 5, 2020.
• [14] R. Rudolph, H. Lilie, “In vitro folding of inclusion body proteins”, Federation of American Societies for Experimental Biology, vol.10, no. 1, pp. 49–56,1996.
• [15] E. D. Clark, “Refolding of recombinant proteins”, Current Opinion in Biotechnology, vol. 9, no.2, 157–163, 1998.
• [16] H. Lilie, E. Schwarz, R. Rudolph, “Advances in refolding of proteins produced in E. coli”, Current Opinion in Biotechnology, vol. 9, no. 5, 497–501,1998.
• [17] Ö. Kaplan, R. Imamoğlu, I. Gökçe, “High-Level Production of MMLV Reverse Transcriptase Enzyme in Escherichia coli”, International Journal of Advances in Engineering and Pure Sciences Accepts, vol. 33, no. 4, pp. 549-555, 2021.
• [18] M. R. Ki, S. P. Pack, “Fusion tags to enhance heterologous protein expression”, Applied Microbiology and Biotechnology, vol. 104, pp. 2411–2425, 2020.
• [19] W. Schumann, L. C. S. Ferreira, “Production of recombinant proteins in Escherichia coli”, Genetics and Molecular Biology, 27, 3, 442-453, 2004.
• [20] D. B. Smith, K. S. Johnson, “Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase”, Gene, vol. 67, pp. 31–40, 1988.
• [21] E. R. La Vallie, E. A. Di Blasio, S. Kovacic, K. L. Grant, “Schendel, P. F.; McCoy, J.M. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm”, Biotechnology, vol. 11, no. 2, pp. 187–193,1993.
• [22] C. V. Maina, P. D. Riggs, A. G. Grandea, B. E. Slatko, L.S. Moran, J. A. Tagliamonte, L. A. McReynolds, C. Di Guan, “An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein”, Gene, vol. 74, no. 2, pp. 365–373, 1988.
• [23] J. G. Marblestone, S. C. Edavettal, Y. Lim, P. Lim, X. Zuo, T.R. Butt, “Comparison of SUMO fusion technology with traditional gene fusion systems: Enhanced expression and solubility with SUMO”, Protein Science, vol. 15, no. 1, pp. 182–189, 2006.
• [24] X. Wu, D. Wu, Z. Lu, W. Chen, X. Hu, Y. Ding, “A Novel Method for High-Level Production of TEV Protease by Superfolder GFP Tag”, Journal of Biomedicine and Biotechnology, 2009.
• [25] J. D. Pedelacq, S. Cabantous, T. Tran, T. C. Terwilliger, G. S. Waldo, “Engineering and characterization of a superfolder green fluorescent protein”, Nature Biotechnology, vol. 24, pp. 79–88, 2006.
• [26] Z. Zhang, R. Tang, D. Zhu, W. Wang, L. Yi, L. Ma, “Non-peptide guided auto-secretion of recombinant proteins by superfolder green fuorescent protein in Escherichia coli”, Scientific Reports, vol. 7, no. 6990, 2017.
• [27] M. Liu, B. Wang, F. Wang, Z. Yang, D. Gao, C. Zhang, L. Ma, X. Yu, “Soluble expression of single-chain variable fragment (scFv) in Escherichia coli using superfolder green fluorescent protein as fusion partner”, Applied Microbiology and Biotechnology, vol.103, pp. 6071–6079, 2019.
• [28] S. Bilgin, Y. Ulusu, H. Kuduğ, I. Gökçe, “Cloning, Expression and Characterization of Xylanase (xyn-akky1) from Bacillus subtilis in Escherichia coli”, Sakarya University Journal of Science, vol. 22, no. 6, pp. 1508–1517, 2018.
• [29] I. Incir, O. Kaplan, S. Bilgin, I Gökçe , “Development of a Fluorescent Protein Based FRET Biosensor for Determination of Protease Activity”, Sakarya University Journal of Science, vol. 25, no. 5, pp. 1235 - 1244, 2021.
• [30] S. Erden Tayhan, S. Bilgin, M. Elmastaş, “Evaluation of the wound healing potential of Teucrioside”, International Journal of Chemistry and Technology, vol. 2, no.1, pp. 16-19, 2018.
• [31] P. R. Mitt, J. P. Priestle, D. A. Cox, G. McMaster, N. Cerletti, M. G. Grütter, “The crystal structure of TGF-beta 3 and comparison to TGF-beta 2: implications for receptor binding”, Protein Science, vol. 5, pp. 1261–1271, 1996.
• [32] H. Abrahamse, M. R. Hamblin, “New photosensitizers for photodynamic therapy”, Biochemical Journal, vol. 473, no. 4, pp. 347–364, 2016.
• [33] A. P. Castanoa, Q. Liua, M. R. Hamblina, “Photodynamic therapy cures green fluorescent protein expressing RIF1 tumors in mice”, Laser Interaction with Tissue and Cells, vol. 5319, pp. 1605-7422.
• [34] V. Nesi-Reis, D. S. S. L. Lera-Nonose, J. Oyama, M. P. P. Silva-Lalucci, I. G. Demarchi, S. M. A. Aristides, J. J. V. Teixeira, T. G. V. Silveira, M. V. C. Lonardoni, “Contribution of photodynamic therapy in wound healing: A systematic review”, Photodiagnosis and Photodynamic Therapy, vol. 21, pp. 294-305, 2018.
• [35] K. Jiang, G. Chun, Z. Wang, Q. Du, A. Wang, Y. Xiong, “Effect of transforming growth factor β3 on the expression of Smad3 and Smad7 in tenocytes”, Molecular Medicine Reports, vo. 13, pp. 3567-3573, 2016.
• [36] A. S. Colwell, Thomas M. Krummel, M. T. Longaker, H. P. Lorenz, “Fetal and Adult Fibroblasts Have Similar TGF-β–Mediated, Smad-Dependent Signaling Pathways”, Plastic and Reconstructive Surgery, vol. 117, no. 7, pp. 2277-2283, 2006.