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Expression and Secretion of Endostar Protein by Escherichia Coli: Optimization of Culture Conditions Using the Response Surface Methodology

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Abstract

Endostar as a specific drug in treatment of the nonsmall cell lung cancer is produced using Escherichia coli expression system. Plackett–Burman design (PBD) and response surface methodology (RSM) are statistical tools for experimental design and optimization of biotechnological processes. This investigation aimed to predict and develop the optimal culture condition and its components for expression and secretion of endostar into the culture medium of E. coli. The synthetic endostar coding sequence was fused with PhoA signal peptide. The nine factors involved in the production of recombinant protein—postinduction temperature, cell density, rotation speed, postinduction time, concentration of glycerol, IPTG, peptone, glycine, and triton X-100—were evaluated using PBD. Four significant factors were selected based on PBD results for optimizing culture condition using RSM. Endostar was purified using cation exchange chromatography and size exclusion chromatography. The maximum level of endostar was obtained under the following condition: 13.57-h postinduction time, 0.76 % glycine, 0.7 % triton X-100, and 4.87 % glycerol. The predicted levels of endostar was significantly correlated with experimental levels (R 2 = 0.982, P = 0.00). The obtained results indicated that PBD and RSM are effective tools for optimization of culture condition and its components for endostar production in E. coli. The most important factors in the enhancement of the protein production are glycerol, glycine, and postinduction time.

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References

  1. Ma, Y., Jin, X.-B., Chu, F.-J., Bao, D.-M., & Zhu, J.-Y. (2014). Expression of liver-targeting peptide modified recombinant human endostatin and preliminary study of its biological activities. Applied Microbiology and Biotechnology, 98(18), 7923–7933.

    Article  CAS  Google Scholar 

  2. Xu, X., Mao, W., Chen, Q., Zhuang, Q., Wang, L., Dai, J., et al. (2014). Endostar, a modified recombinant human endostatin, suppresses angiogenesis through inhibition of Wnt/β-catenin signaling pathway. PLoS One, 9(9), e107463.

    Article  Google Scholar 

  3. Su, Z., Wu, X., Feng, Y., Ding, C., Xiao, Y., Cai, L., et al. (2007). High level expression of human endostatin in Pichia pastoris using a synthetic gene construct. Applied Microbiology and Biotechnology, 73(6), 1355–1362.

    Article  CAS  Google Scholar 

  4. Du, C., Yi, X., & Zhang, Y. (2010). Expression and purification of soluble recombinant human endostatin in Escherichia coli. Biotechnology and Bioprocess Engineering, 15(2), 229–235.

    Article  CAS  Google Scholar 

  5. Mohajeri, A., Pilehvar-soltanahmadi, Y., Abdolalizadeh, J., Karimi, P., Zarghami, N. (2016). Effect of Culture Condition Variables on Human Endostatin Gene Expression in Escherichia coli Using Response Surface Methodology. Jundishapur Journal of Microbiology, (In Press), e34091.

  6. Ameghi, A., Baradaran, B., Aghaiypour, K., Barzegar, A., Pilehvar-Soltanahmadi, Y., Moghadampour, M., et al. (2015). Generation of new M2e-HA2 fusion chimeric peptide to development of a recombinant fusion protein vaccine. Advanced pharmaceutical bulletin, 5(Suppl 1), 673.

    Article  Google Scholar 

  7. Niri, N. M., Memarnejadian, A., Hadjati, J., Aghasadeghi, M. R., Shokri, M., Pilehvar-soltanahmadi, Y., et al. (2016). Construction and production of Foxp3-Fc (IgG) DNA vaccine/fusion protein. Avicenna journal of medical biotechnology, 8(2), 57.

    Google Scholar 

  8. Singh, A., Upadhyay, V., Upadhyay, A. K., Singh, S. M., & Panda, A. K. (2015). Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. Microbial Cell Factories, 14(1), 41.

    Article  Google Scholar 

  9. Mohajeri, A., Pilehvar-Soltanahmadi, Y., Pourhassan-Moghaddam, M., Abdolalizadeh, J., Karimi, P., & Zarghami, N. (2016). Cloning and expression of recombinant human endostatin in periplasm of Escherichia coli expression system. Advanced Pharmaceutical Bulletin, 6(2), 187–194.

    Article  Google Scholar 

  10. Huang, X., Wong, M. K., Zhao, Q., Zhu, Z., Wang, K. Z., Huang, N., et al. (2001). Soluble recombinant endostatin purified from Escherichia coli: antiangiogenic activity and antitumor effect. Cancer Research, 61(2), 478–481.

    CAS  Google Scholar 

  11. Xu, R., Du, P., Fan, J.-J., Zhang, Q., Li, T.-P., & Gan, R.-B. (2002). High-level expression and secretion of recombinant mouse endostatin by Escherichia coli. Protein Expression and Purification, 24(3), 453–459.

    Article  CAS  Google Scholar 

  12. Vaz, M. R. F., de Sousa Junior, F. C., Costa, L. M. R., dos Santos, E. S., Martins, D. R. A., & de Macedo, G. R. (2015). Optimization of culture medium for cell growth and expression of 648 antigen from Leishmania infantum chagasi in recombinant Escherichia coli M15. Annals of Microbiology, 65(3), 1607–1613.

    Article  CAS  Google Scholar 

  13. Maharjan, S., Singh, B., Bok, J.-D., Kim, J.-I., Jiang, T., Cho, C.-S., et al. (2014). Exploring Codon Optimization and Response Surface Methodology to Express Biologically Active Transmembrane RANKL in E. coli. PLoS One, 9(5), e96259.

    Article  Google Scholar 

  14. Lee, M. S., Lin, I. F., Lai, G. H., Lin, Y. C., & Li, K. Y. (2015). Statistical optimization of culture medium for the overproduction of chicken anemia virus immunogen-VP1 protein in a recombinant E. coli for vaccine application. Asia-Pacific Journal of Chemical Engineering, 10(1), 96–104.

    Article  CAS  Google Scholar 

  15. Yari, K., Afzali, S., Mozafari, H., Mansouri, K., & Mostafaie, A. (2013). Molecular cloning, expression and purification of recombinant soluble mouse endostatin as an anti-angiogenic protein in Escherichia coli. Molecular Biology Reports, 40(2), 1027–1033.

    Article  CAS  Google Scholar 

  16. Cao, W., Li, H., Zhang, J., Li, D., Acheampong, D. O., Chen, Z., & Wang, M. (2013). Periplasmic expression optimization of VEGFR2 D3 adopting response surface methodology: antiangiogenic activity study. Protein Expression and Purification, 90(2), 55–66.

    Article  CAS  Google Scholar 

  17. Wu, J., Fu, W., Luo, J., & Zhang, T. (2005). Expression and purification of human endostatin from Hansenula polymorpha A16. Protein Expression and Purification, 42(1), 12–19.

    Article  Google Scholar 

  18. Papaneophytou, C., & Kontopidis, G. (2016). A comparison of statistical approaches used for the optimization of soluble protein expression in Escherichia coli. Protein Expression and Purification, 120, 126–137.

    Article  CAS  Google Scholar 

  19. Dilipkumar, M., Rajasimman, M., & Rajamohan, N. (2011). Response surface methodology for the optimization of inulinase production by K. marxianus var. marxianus. Journal of Applied Sciences in Environmental Sanitation, 6(1), 85–95.

    CAS  Google Scholar 

  20. Kahyaoglu, T. (2008). Optimization of the pistachio nut roasting process using response surface methodology and gene expression programming. LWT-Food Science and Technology, 41(1), 26–33.

    Article  CAS  Google Scholar 

  21. Al-Samarrai, T. H., Jones, W. T., Harvey, D., Kirk, C. A., & Templtone, M. (2013). Effect of 4% glycerol and low aeration on result of expression in Escherichia coli of Cin3 and three Venturia inaequalis EST’s recombinant proteins. American Journal of Molecular Biology, 3, 1–9.

    Article  CAS  Google Scholar 

  22. X-b, D. U., Ye, S., Feng, L., K-y, Z. H. E. N. G., K-w, W. A. N. G., T-t, L. I. N., et al. (2007). Chemical chaperones increasing expression level of soluble single-chain Fv antibody (scFv2F3). Chemical Research in Chinese Universities, 23(1), 69–75.

    Article  Google Scholar 

  23. Gekko, K., & Timasheff, S. N. (1981). Mechanism of protein stabilization by glycerol: preferential hydration in glycerol-water mixtures. Biochemistry, 20(16), 4667–4676.

    Article  CAS  Google Scholar 

  24. Choi, J., & Lee, S. (2004). Secretory and extracellular production of recombinant proteins using Escherichia coli. Applied Microbiology and Biotechnology, 64(5), 625–635.

    Article  CAS  Google Scholar 

  25. Li, B., Wang, L., Su, L., Chen, S., Li, Z., Chen, J., & Wu, J. (2012). Glycine and Triton X-100 enhanced secretion of recombinant α-CGTase mediated by OmpA signal peptide in Escherichia coli. Biotechnology and Bioprocess Engineering, 17(6), 1128–1134.

    Article  CAS  Google Scholar 

  26. Manderson, D., Dempster, R., & Chisti, Y. (2006). Production of an active recombinant Aspin antigen in Escherichia coli for identifying animals resistant to nematode infection. Enzyme and microbial technology, 38(5), 591–598.

    Article  CAS  Google Scholar 

  27. Wei, D. M., Gao, Y., Cao, X. R., Zhu, N. C., Liang, J. F., Xie, W. P., et al. (2006). Soluble multimer of recombinant endostatin expressed in E. coli has anti-angiogenesis activity. Biochemical and biophysical research communications, 345(4), 1398–1404.

    Article  CAS  Google Scholar 

  28. Akbari, H., Jafarian-Dehkordi, A., Chou, C. P., & Abedi, D. V. (2014). Optimization of a single-chain antibody fragment overexpression in Escherichia coli using response surface methodology. Research in Pharmaceutical Sciences, 10(1), 75–83.

    Google Scholar 

  29. Larentis, A. L., Nicolau, J. F., dos Esteves, G., Vareschini, D. T., de Almeida, F. V., dos Reis, M. G., et al. (2014). Evaluation of pre-induction temperature, cell growth at induction and IPTG concentration on the expression of a leptospiral protein in E. coli using shaking flasks and microbioreactor. BMC research notes, 7(1), 671.

    Article  Google Scholar 

  30. Papaneophytou, C. P., & Kontopidis, G. (2014). Statistical approaches to maximize recombinant protein expression in Escherichia coli: a general review. Protein Expression and Purification, 94, 22–32.

    Article  CAS  Google Scholar 

  31. Hernández, V. E. B., Maldonado, L. M. P., Rivero, E. M., de la Rosa, A. P. B., Acevedo, L. G. O., & Rodríguez, A. D. L. (2008). Optimization of human interferon gamma production in Escherichia coli by response surface methodology. Biotechnology and Bioprocess Engineering, 13(1), 7–13.

    Article  Google Scholar 

  32. Vera, A., González-Montalbán, N., Arís, A., & Villaverde, A. (2007). The conformational quality of insoluble recombinant proteins is enhanced at low growth temperatures. Biotechnology and Bioengineering, 96(6), 1101–1106.

    Article  CAS  Google Scholar 

  33. Wang, Yh, Jing, Cf, Yang, B., Mainda, G., Dong, Ml, & Xu, Al. (2005). Production of a new sea anemone neurotoxin by recombinant Escherichia coli: optimization of culture conditions using response surface methodology. Process Biochemistry, 40(8), 2721–2728.

    Article  CAS  Google Scholar 

  34. Hartmann, C., & Engel, A. (2011). Cloning, expression, purification, and characterization of the membrane protein UncI from Escherichia coli. Protein Expression and Purification, 79(2), 187–190.

    Article  CAS  Google Scholar 

  35. DeLisa, M., Chae, H., Weigand, W., Valdes, J., Rao, G., & Bentley, W. (2001). Generic model control of induced protein expression in high cell density cultivation of Escherichia coli using on-line GFP-fusion monitoring. Bioprocess and Biosystems Engineering, 24(2), 83–91.

    Article  CAS  Google Scholar 

  36. Lim, H. K., & Jung, K. H. (1998). Improvement of heterologous protein productivity by controlling postinduction specific growth rate in recombinant Escherichia coli under control of the PL promoter. Biotechnology Progress, 14(4), 548–553.

    Article  CAS  Google Scholar 

  37. Ranjbari, J., Babaeipour, V., Vahidi, H., Moghimi, H., Mofid, M., Namvaran, M., & Jafari, S. (2015). Enhanced Production of Insulin-Like Growth Factor I Protein in Escherichia coli by optimization of five key factors. Iranian Journal of Pharmaceutical Research, 14(3), 907–917.

    Google Scholar 

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Acknowledgments

The authors are so thankful to their colleagues at Tuberculosis and Lung Disease Research Center of Tabriz University of Medical Sciences. The authors are thankful to their coworkers in the Drug Applied Research Center of Tabriz University of Medical Sciences. We thank Dr. Sarvin Sanyi for assistance in editing this manuscript.

Authors’ contributions

This work was a part of Ph.D. thesis of Abbas Mohajeri and he wrote the paper. Nosratollah Zarghami was the supervisor of the thesis that edited the manuscript, directed the research, and coordinated the study. Abbas Mohajeri performed plasmid design and molecular works, carried out the analysis, and wrote the manuscript draft. Younes Pilehvar-Soltanahmadi participated in practical activities. Jalal Abdolalizadeh and Farhad Kiafar participated as technical assistance advisor. All the authors read and approved the final manuscript.

Funding/Support

This research was financially supported by a grant (Project Number 5/76/520) from the Tuberculosis and Lung Disease Research Center, Tabriz University of Medical Sciences. Tabriz, Iran.

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Authors and Affiliations

  1. Tuberculosis and Lung Disease Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

    Abbas Mohajeri, Younes Pilehvar-Soltanahmadi & Nosratollah Zarghami

  2. Department of Biotechnology, Zahravi pharmaceutical company, Tabriz, Iran

    Abbas Mohajeri & Farhad Kiafar

  3. Drug Applied Research Center, Immunology Lab, Tabriz University of Medical Sciences, Tabriz, Iran

    Jalal Abdolalizadeh & Nosratollah Zarghami

  4. Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran

    Younes Pilehvar-Soltanahmadi & Nosratollah Zarghami

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  1. Abbas Mohajeri
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  2. Jalal Abdolalizadeh
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Correspondence to Nosratollah Zarghami.

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Mohajeri, A., Abdolalizadeh, J., Pilehvar-Soltanahmadi, Y. et al. Expression and Secretion of Endostar Protein by Escherichia Coli: Optimization of Culture Conditions Using the Response Surface Methodology. Mol Biotechnol 58, 634–647 (2016). https://doi.org/10.1007/s12033-016-9963-9

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  • DOI: https://doi.org/10.1007/s12033-016-9963-9

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