Skip to main content
SpringerLink
Log in

Impacts of Magnetic Immobilization on the Recombinant Proteins Structure Produced in Pichia pastoris System

  • Original Paper
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Pichia pastoris expression system was introduced with post-translation process similar to higher eukaryotes. Preliminary studies were performed toward process intensification and magnetic immobilization of this system. In this experiment, effects of magnetic immobilization on the structure of recombinant protein were evaluated. P. pastoris cell which express human serum albumin (HSA) was used as a model. The cells were immobilized with various concentrations of APTES coated magnetite nanoparticles. HSA production was done over 5 days induction and structure of the product was analyzed by UV–vis, fluorescence, and ATR-FTIR spectroscopy. Second derivative deconvolution method was used to analyze the secondary structure of HSA. P. pastoris cell that were immobilized with 0.5 and 1 mg/mL of nanoparticles were produced HSA with intact structure. But immobilization with 2 mg/mL of nanoparticles resulted in some modifications in the secondary structures (i.e., α-helixes and β-turns) of produced HSA. Based on these data, immobilization of P. pastoris cells with 0.5 or 1 mg/mL of nanoparticles is completely efficient for cell harvesting and has any effect on the structure of recombinant product. These findings revealed that decoration of microbial cells with high concentrations of nanoparticles has some impacts on the structure of secretory proteins.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Scheme 1
Scheme 2

Similar content being viewed by others

References

  1. Lu, J., Peng, W., Lv, Y., Jiang, Y., Xu, B., Zhang, W., et al. (2020). Application of cell immobilization technology in microbial cocultivation systems for biochemicals production. Industrial and Engineering Chemistry Research, 59, 17026–17034.

    Article  CAS  Google Scholar 

  2. Bouabidi, Z. B., El-Naas, M. H., & Zhang, Z. (2019). Immobilization of microbial cells for the biotreatment of wastewater: A review. Environmental Chemistry Letters, 17, 241–257.

    Article  CAS  Google Scholar 

  3. Trelles, J. A., & Rivero, C. W. (2020). Whole cell entrapment techniques. In J. M. Guisan, J. Bolivar, F. López-Gallego, & J. Rocha-Martín (Eds.), Immobilization of enzymes and cells (Vol. 2100, pp. 385–394). New York: Humana.

    Chapter  Google Scholar 

  4. Abarian, M., Hassanshahian, M., & Esbah, A. (2019). Degradation of phenol at high concentrations using immobilization of Pseudomonas putida P53 into sawdust entrapped in sodium-alginate beads. Water Science and Technology, 79, 1387–1396.

    Article  CAS  Google Scholar 

  5. Terpou, A., Ganatsios, V., Kanellaki, M., & Koutinas, A. A. (2020). Entrapped psychrotolerant yeast cells within pine sawdust for low temperature wine making: Impact on wine quality. Microorganisms, 8, 764.

    Article  CAS  Google Scholar 

  6. Chen, C., Hu, J., & Wang, J. (2020). Uranium biosorption by immobilized active yeast cells entrapped in calcium-alginate-PVA-GO-crosslinked gel beads. Radiochimica Acta, 108, 273–286.

    Article  CAS  Google Scholar 

  7. Göksungur, Y., & Zorlu, N. (2001). Production of ethanol from beet molasses by Ca-alginate immobilized yeast cells in a packed-bed bioreactor. Turk. J. Biol., 25, 265–275.

    Google Scholar 

  8. Thipayarat, A. (2003). Production of human serum albumin by immobilized yeast. Ph.D. Thesis, Syracuse University, Syracuse, USA.

  9. Berovic, M., Berlot, M., Kralj, S., & Makovec, D. (2014). A new method for the rapid separation of magnetized yeast in sparkling wine. Biochemical Engineering Journal, 88, 77–84.

    Article  CAS  Google Scholar 

  10. Safarik, I., & Safarikova, M. (2007). Magnetically modified microbial cells: A new type of magnetic adsorbents. China Particuology, 5, 19–25.

    Article  CAS  Google Scholar 

  11. Safarik, I., Maderova, Z., Pospiskova, K., Baldikova, E., Horska, K., & Safarikova, M. (2015). Magnetically responsive yeast cells: Methods of preparation and applications. Yeast, 32, 227–237.

    CAS  PubMed  Google Scholar 

  12. Ebrahiminezhad, A., Taghizadeh, S.-M., Ghasemi, Y., & Berenjian, A. (2020). Immobilization of cells by magnetic nanoparticles. In J. M. Guisan, J. M. Bolivar, F. López-Gallego, & J. Rocha-Martín (Eds.), Immobilization of enzymes and cells (Vol. 2100, pp. 427–435). New York: Humana.

    Chapter  Google Scholar 

  13. Safarik, I., Pospiskova, K., Maderova, Z., Baldikova, E., Horska, K., & Safarikova, M. (2015). Microwave-synthesized magnetic chitosan microparticles for the immobilization of yeast cells. Yeast, 32, 239–243.

    CAS  PubMed  Google Scholar 

  14. Ansari, F., Grigoriev, P., Libor, S., Tothill, I. E., & Ramsden, J. J. (2009). DBT degradation enhancement by decorating Rhodococcus erythropolis IGST8 with magnetic Fe3O4 nanoparticles. Biotechnology and Bioengineering, 102, 1505–1512.

    Article  CAS  Google Scholar 

  15. Raee, M. J., Ebrahiminezhad, A., Gholami, A., Ghoshoon, M. B., & Ghasemi, Y. (2018). Magnetic immobilization of recombinant E. coli producing extracellular asparaginase: An effective way to intensify downstream process. Separation Science and Technology, 53, 1397–1404.

    Article  CAS  Google Scholar 

  16. Taghizadeh, S.-M., Jafari, S., Ahmad-Kiadaliri, T., Mobasher, M. A., Lal, N., Raee, M. J., et al. (2019). Magnetic immobilisation as a promising approach against bacteriophage infection. Materials Research Express, 6, 1250–1258.

    Google Scholar 

  17. Taghizadeh, S.-M., Ebrahiminezhad, A., Ghoshoon, M. B., Dehshahri, A., Berenjian, A., & Ghasemi, Y. (2020). Magnetic immobilization of Pichia pastoris cells for the production of recombinant human serum albumin. Nanomaterials, 10, 111.

    Article  CAS  Google Scholar 

  18. Ebrahiminezhad, A., Varma, V., Yang, S., & Berenjian, A. (2016). Magnetic immobilization of Bacillus subtilis natto cells for menaquinone-7 fermentation. Applied Microbiology and Biotechnology, 100, 173–180.

    Article  CAS  Google Scholar 

  19. Ebrahiminezhad, A., Varma, V., Yang, S., Ghasemi, Y., & Berenjian, A. (2015). Synthesis and application of amine functionalized iron oxide nanoparticles on menaquinone-7 fermentation: A step towards process intensification. Nanomaterials, 6, 1.

    Article  Google Scholar 

  20. Taghizadeh, S. M., Berenjian, A., Chew, K. W., Show, P. L., Mohd Zaid, H. F., Ramezani, H., et al. (2020). Impact of magnetic immobilization on the cell physiology of green unicellular algae Chlorella vulgaris. Bioengineered, 11, 141–153.

    Article  CAS  Google Scholar 

  21. Daly, R., & Hearn, M. T. (2005). Expression of heterologous proteins in Pichia pastoris: A useful experimental tool in protein engineering and production. Journal of Molecular Recognition, 18, 119–138.

    Article  CAS  Google Scholar 

  22. Lima-Pérez, J., Rodríguez-Gómez, D., Loera, O., Viniegra-González, G., & López-Pérez, M. (2018). Differences in growth physiology and aggregation of Pichia pastoris cells between solid-state and submerged fermentations under aerobic conditions. Journal of Chemical Technology and Biotechnology, 93, 527–532.

    Article  Google Scholar 

  23. Doaga, R., McCormac, T., & Dempsey, E. (2020). Functionalized magnetic nanomaterials for electrochemical biosensing of cholesterol and cholesteryl palmitate. Microchimica Acta, 187, 225.

    Article  CAS  Google Scholar 

  24. Kaushik, N., Lamminmäki, U., Khanna, N., & Batra, G. (2020). Enhanced cell density cultivation and rapid expression-screening of recombinant Pichia pastoris clones in microscale. Scientific Reports, 10, 7458.

    Article  CAS  Google Scholar 

  25. Olson, B. J., & Markwell, J. (2007). Assays for determination of protein concentration (p. 48). Pharmacol: Curr. Protoc.

    Google Scholar 

  26. Yang, Q., Liang, J., & Han, H. (2009). Probing the interaction of magnetic iron oxide nanoparticles with bovine serum albumin by spectroscopic techniques. The Journal of Physical Chemistry B, 113, 10454–10458.

    Article  CAS  Google Scholar 

  27. Usoltsev, D., Sitnikova, V., Kajava, A., & Uspenskaya, M. (2019). Systematic FTIR spectroscopy study of the secondary structure changes in human serum albumin under various denaturation conditions. Biomolecules, 9, 359.

    Article  CAS  Google Scholar 

  28. Ebrahiminezhad, A., Ghasemi, Y., Rasoul-Amini, S., Barar, J., & Davaran, S. (2013). Preparation of novel magnetic fluorescent nanoparticles using amino acids. Colloids and Surfaces B, 102, 534–539.

    Article  CAS  Google Scholar 

  29. Rosenholm, J. M., Zhang, J., Sun, W., & Gu, H. (2011). Large-pore mesoporous silica-coated magnetite core-shell nanocomposites and their relevance for biomedical applications. Microporous and Mesoporous Materials, 145, 14–20.

    Article  CAS  Google Scholar 

  30. Pham, X. N., Nguyen, T. P., Pham, T. N., Tran, T. T. N., & Tran, T. V. T. (2016). Synthesis and characterization of chitosan-coated magnetite nanoparticles and their application in curcumin drug delivery. Advances in Natural Sciences: Nanoscience and Nanotechnology, 7, 045010.

    Google Scholar 

  31. Li, Y. G., Gao, H. S., Li, W. L., Xing, J. M., & Liu, H. Z. (2009). In situ magnetic separation and immobilization of dibenzothiophene-desulfurizing bacteria. Bioresource Technology, 100, 5092–5096.

    Article  CAS  Google Scholar 

  32. Aghili, Z., Taheri, S., Zeinabad, H. A., Pishkar, L., Saboury, A. A., Rahimi, A., et al. (2016). Investigating the interaction of Fe nanoparticles with lysozyme by biophysical and molecular docking studies. PLoS ONE, 11, e0164878.

    Article  Google Scholar 

  33. Varkhede, N., Peters, B.-H., Wei, Y., Middaugh, C. R., Schöneich, C., & Forrest, M. L. (2019). Effect of iron oxide nanoparticles on the oxidation and secondary structure of growth hormone. Journal of Pharmaceutical Sciences, 108, 3372–3381.

    Article  CAS  Google Scholar 

  34. Vakili-Ghartavol, R., Momtazi-Borojeni, A. A., Vakili-Ghartavol, Z., Aiyelabegan, H. T., Jaafari, M. R., Rezayat, S. M., et al. (2020). Toxicity assessment of superparamagnetic iron oxide nanoparticles in different tissues. Artificial Cells, Nanomedicine, and Biotechnology, 48, 443–451.

    Article  CAS  Google Scholar 

  35. Malhotra, N., Lee, J.-S., Liman, R. A. D., Ruallo, J. M. S., Villaflores, O. B., Ger, T.-R., et al. (2020). Potential toxicity of iron oxide magnetic nanoparticles: A review. Molecules, 25, 3159.

    Article  CAS  Google Scholar 

  36. Simonsen, G., Strand, M., & Øye, G. (2018). Potential applications of magnetic nanoparticles within separation in the petroleum industry. Journal of Petroleum Science and Engineering, 165, 488–495.

    Article  CAS  Google Scholar 

  37. Antarnusa, G., & Suharyadi, E. (2020). A synthesis of polyethylene glycol (PEG)-coated magnetite Fe3O4 nanoparticles and their characteristics for enhancement of biosensor. Materials Research Express, 7, 056103.

    Article  CAS  Google Scholar 

  38. Lazaro-Carrillo, A., Filice, M., Guillén, M. J., Amaro, R., Viñambres, M., Tabero, A., et al. (2020). Tailor-made PEG coated iron oxide nanoparticles as contrast agents for long lasting magnetic resonance molecular imaging of solid cancers. Materials Science and Engineering C, 107, 110262.

    Article  CAS  Google Scholar 

  39. Pham, X.-H., Hahm, E., Kim, H.-M., Son, B. S., Jo, A., An, J., et al. (2020). Silica-coated magnetic iron oxide nanoparticles grafted onto graphene oxide for protein isolation. Nanomaterials, 10, 117.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This experiment was financially supported by Shiraz University of Medical Sciences as PhD thesis proposal of Seyedeh-Masoumeh Taghizadeh submitted at No. 18588 in the School of Pharmacy. Authors are grateful to the support provided by the University of Waikato, New Zealand.

Author information

Authors and Affiliations

  1. Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran

    Seyedeh-Masoumeh Taghizadeh, Younes Ghasemi & Ali Dehshahri

  2. Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

    Seyedeh-Masoumeh Taghizadeh, Mohammad Bagher Ghoshoon & Younes Ghasemi

  3. Biotechnology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

    Mohammad Bagher Ghoshoon

  4. School of Engineering, Faculty of Science and Engineering, The University of Waikato, Hamilton, 3240, New Zealand

    Aydin Berenjian

  5. Department of Medical Nanotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Unit 23, 2nd Floor, Almass Building, Alley 29, Ghasrodasht St., Shiraz, Iran

    Alireza Ebrahiminezhad

Authors
  1. Seyedeh-Masoumeh Taghizadeh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mohammad Bagher Ghoshoon
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Aydin Berenjian
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Younes Ghasemi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ali Dehshahri
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Alireza Ebrahiminezhad
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Aydin Berenjian or Alireza Ebrahiminezhad.

Ethics declarations

Conflict of interest

The authors declare that there is no conflict of interest with the content of this article.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Taghizadeh, SM., Ghoshoon, M.B., Berenjian, A. et al. Impacts of Magnetic Immobilization on the Recombinant Proteins Structure Produced in Pichia pastoris System. Mol Biotechnol 63, 80–89 (2021). https://doi.org/10.1007/s12033-020-00286-4

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-020-00286-4

Keywords

Search

Navigation