Skip to main content
SpringerLink
Log in

Expression and Control of Codon-Optimized Granulocyte Colony-Stimulating Factor in Pichia pastoris

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Granulocyte colony-stimulating factor (GCSF) has therapeutic applications due to its proven efficacy in different forms of neutropenia and chemotherapy-induced leucopenia. The original 564-bp nucleotide sequence from NCBI was codon optimized and assembled by overlapping PCR method comprising of 16 oligos of 50-nt length with 15 base overhang. The synthetic gene (CO-GCSF) was cloned under glucose utilizing glyceraldehyde 3-phosphate dehydrogenase (GAP) and methanol-utilizing alcohol oxidase (AOX1) promoters and expressed in Pichia pastoris SMD1168 strain. Constitutive expression under GAP resulted in cellular toxicity while AOX1 promoter controlled expression was stable. Variation in the levels of expression was observed among the transformant colonies with transformant #2 secreting up to ∼4 mg/L of GCSF. The molecular mass of the expressed GCSF in P. pastoris was ∼19.0 kDa. Quatitation of the expressed protein was carried out by a highly reproducible gel densitometric method. Effect of several operational and nutritional conditions was studied on GCSF production and the results suggest a general approach for increasing the yield of GCSF several folds (2- to 5-fold) over the standard conditions employed currently. Cultivation of the single-copy integrant in the chemically defined medium in a 5-L fermenter resulted in a volumetric productivity of ∼0.7 mg/L/h at the end of the induction phase, which was about 4-fold higher than attained in the shake flask.

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

Similar content being viewed by others

References

  1. Biethahna, S., Alvesa, F., Wildea, S., Hiddemanna, W., & Spiekermann, K. (1999). Expression of granulocyte colony-stimulating factor and granulocyte-macrophage colony stimulating factor associated signal transduction proteins of the JAK/STAT pathway in normal granulopoiesis and in blast cells of acute myelogenous leukemia. Experimental Hematology, 27, 885–894.

    Article  Google Scholar 

  2. Viret, F., Goncalves, A., Tarpin, C., Chabannon, C., et al. (2006). GCSF in oncology. Bulletins Cancer, 93, 463–471.

    CAS  Google Scholar 

  3. Bell, E. (2009). Transplantation: GCSF therapy after BMT: getting the timing right. Nature Reviews in Immunology, 9, 308–309.

    Article  CAS  Google Scholar 

  4. Nagata, S., Tsuchiya, M., Asano, S., Yamamoto, O., Hirata, Y., Kubota, N., Oheda, M., Nomura, H., & Yamazaki, T. (1986). The chromosomal gene structure and two mRNAs for human granulocyte colony stimulating factor. EMBO Journal, 5, 575–581.

    CAS  Google Scholar 

  5. Kubota, N., Orita, T., Hattori, K., Oh-eda, M., et al. (1990). Structural characterization of natural and recombinant human granulocyte colony-stimulating factors. Journal of Biochemistry, 107, 486–492.

    CAS  Google Scholar 

  6. Oh-eda, M., Hasegawa, M., Hattori, K., Kuboniwa, H., et al. (1990). O-linked sugar chain of human granulocyte colony-stimulating factor protects it against polymerization and denaturation allowing it to retain its biological activity. Journal of Biological Chemistry, 265, 11432–11435.

    CAS  Google Scholar 

  7. Carter, C. R. D., Whitmore, K. M., & Thorpe, R. (2004). The significance of carbohydrates on G-CSF: differential sensitivity of GCSFs’ to human neutrophil elastase degradation. Journal of Leukocyte Biology, 75, 515–522.

    Article  CAS  Google Scholar 

  8. Maccani, A., Landes, N., Stadlmayr, G., Maresch, D., Leitner, C., Maurer, M., Gasser, B., Ernst, W., Kunert, R., & Mattanovich, D. (2014). Pichia pastoris secretes recombinant proteins less efficiently than Chinese hamster ovary cells but allows higher space-time yields for less complex proteins. Biotechnology Journal, 9, 526–537.

    Article  CAS  Google Scholar 

  9. Werten, M. W. T., Wisselink, W. H., Jansen-van den Bosch, T. J., de Bruin, E. C., et al. (2001). Secreted production of a custom-designed, highly hydrophilic gelatin in Pichia pastoris. Protein Engineering, 14, 447–454.

    Article  CAS  Google Scholar 

  10. Trinh, L. B., Phue, J. N., & Shiloach, J. (2003). Effect of methanol feeding strategies on production and yield of recombinant mouse endostatin from Pichia pastoris. Biotechnology and Bioengineering, 82, 438–444.

    Article  CAS  Google Scholar 

  11. Fickers, P. (2014). Pichia pastoris: a workhorse for recombinant protein production. Current research in Microbiology and Biotechnology, 2, 354–363.

    Google Scholar 

  12. Saeedinia, A., Shamsara, M., Bahrami, A., Zeinoddini, M., et al. (2008). Heterologous expression of human granulocyte colony stimulating factor in Pichia pastoris. Biotechnology, 2, 1–5.

    Google Scholar 

  13. Bahrami, A., Shojaosadati, S. A., Khalilzadeh, R., Mohammadian, J., Farahani, E. V., & Masoumian, M. R. (2009). Prevention of human granulocyte colony-stimulating factor protein aggregation in recombinant Pichia pastoris fed-batch fermentation using additives. Biotechnology and Applied Biochemistry, 52, 141–148.

    Article  CAS  Google Scholar 

  14. Lasnik, M. A., Porekar, V. G., & Stalc', A. (2001). Human granulocyte colony stimulating factor (hG-CSF) expressed by methylotrophic yeast Pichia pastoris. Pfliigers Archives of European Journal of Physiology, 442, R184–R186.

    Article  CAS  Google Scholar 

  15. Apte-Deshpande, A., Somani, S., Mandal, G., Soorapaneni, S., & Padmanabhan, S. (2009). Over expression and analysis of O-glycosylated recombinant human granulocyte colony stimulating factor in Pichia pastoris using Agilent 2100 Bioanalyzer. Journal of Biotechnology, 143, 44–50.

    Article  CAS  Google Scholar 

  16. Angov, E. (2011). Codon usage: nature’s roadmap to expression and folding of protein. Biotechnology Journal, 6, 650–659.

    Article  CAS  Google Scholar 

  17. Macauley-Patrick, S., Fazenda, M. L., McNeil, B., & Harvey, L. M. (2005). Heterologous protein production using the Pichia pastoris expression system. Yeast, 22, 249–270.

    Article  CAS  Google Scholar 

  18. Pal, Y., Khusboo, A., & Mukherjee, K. J. (2006). Process optimization of constitutive human granulocyte macrophage colony-stimulating factor (hGM-CSF) expression in Pichia pastoris fed batch culture. Applied Microbiology and Biotechnology, 69, 650–657.

    Article  CAS  Google Scholar 

  19. Vogl, T., & Glieder, A. (2013). Regulation of Pichia pastoris promoters and its consequences for protein production. Nature Biotechnology, 30, 385–404.

    CAS  Google Scholar 

  20. Viader-Salvado, J. M., Cab-Barrera, E. L., Galan-Wong, L. J., & Guerrero-Olazaran, M. (2006). Genotyping of recombinant Pichia pastoris strains. Cellular and Molecular Biology Letters, 11, 348–359.

    Article  CAS  Google Scholar 

  21. Jafari, R., Sundstrom, B. E., & Holm, P. (2011). Optimization of production of the anti keratin 8 single chain Fv TS-218 in Pichia pastoris using design of experiments. Microbial Cell Factories, 10, 34.

    Article  Google Scholar 

  22. Jahic, M., Wallberg, F., Bollock, M., Garcia, P., & Enfors, S. O. (2003). Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris bioreactor cultures. Microbial Cell Factories, 2, 6.

    Article  Google Scholar 

  23. André, N., Cherouati, N., Prual, C., Steffan, T., Zeder-Lutz, G., Magnin, T., Pattus, F., Michel, H., Wagner, R., & Reinhart, C. (2006). Enhancing functional production of G protein-coupled receptors in Pichia pastoris to levels required for structural studies via a single expression system. Protein Science, 15, 1115–1126.

    Article  Google Scholar 

  24. Batra, J., Beri, D., & Mishra, S. (2014). Response surface methodology based optimization of β–glucosidase production from Pichia pastoris. Applied Biochemistry and Biotechnology, 172, 380–393.

    Article  CAS  Google Scholar 

  25. Athmaram, T. N., Singh, A. K., Saraswat, S., Srivastava, S., Miisra, P., Rao, M. K., Gopalan, N., & Rao, P. V. L. (2013). A simple Pichia pastoris fermentation and downstream processing strategy for making recombinant pandemic swine origin influenza A virus hemagglutinin protein. Journal of Industrial Microbiology and Biotechnology, 40, 245–255.

    Article  CAS  Google Scholar 

  26. Brierley, R. A., Davis, G. R., Holtz, G.. C., Gleeson, M. A., Howard, B. D. (1997) Production of insulin-like growth factor-1 in methylotropic yeast cells. Google Patents, US 08/308, 196.

  27. Werten, M. W. T., Wisselink, W. H., Jansen-van Den Bosch, T. J. C., de Bruin, E. A., & de Wolf, F. (2001). Secreted production of a custom-designed, highly hydrophilic gelatin in Pichia pastoris. Protein Engineering, 14, 447–454.

    Article  CAS  Google Scholar 

  28. Ricci, M. S., Sarkar, C. A., Fallon, E. M., Lauffenburger, D. A., & Brems, D. N. (2003). pH dependence of structural stability of interleukin-2 and granulocyte colony-stimulating factor. Protein Science, 12, 1030–38.

    Article  Google Scholar 

  29. Gorgens, J. F., van Zyl, W. H., Knoetze, J. H., & Hahn-Hagerdal, B. (2000). Amino acid supplementation improves heterologous protein production by Saccharomyces cerevisiae in defined medium. Applied Microbiology and Biotechnology, 67, 684–691.

    Article  Google Scholar 

  30. Heyland, J., Fu, J., Blank, L. M., & Schmid, A. (2011). Carbon metabolism limits recombinant protein production in Pichia pastoris. Biotechnology and Bioengineering, 108, 1942–1953.

    Article  CAS  Google Scholar 

  31. Ramon, R., Ferrer, P., & Valero, F. (2007). Sorbitol co-feeding reduces metabolic burden caused by the overexpression of Rhizopus oryzae lipase in Pichia pastoris. Journal of Biotechnology, 130, 39–46.

    Article  CAS  Google Scholar 

  32. Cos, O., Ramon, R., Montesinos, J. L., & Valero, F. (2006). Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: a review. Microbial Cell Factories, 5, 17.

    Article  Google Scholar 

  33. Maurer, M., Kuhleitner, M., Gasser, B., & Mattanovich, D. (2006). Versatile modeling and optimization of fed batch processes for the production of secreted heterologous proteins with Pichia pastoris. Microbial Cell Factories, 5, 37.

    Article  Google Scholar 

  34. Werner, R. J. (2004). Economic aspects of commercial manufacture of biopharmaceuticals. Journal of Biotechnology, 113, 171–182.

    Article  CAS  Google Scholar 

  35. Chien, S.-F. (2010). Cloning and expression of bioactive human granulocyte colony stimulating factor in Pichia pastoris. Journal of Chinese Chemical Society, 57, 850–856.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors wish to thank IIT Delhi for providing funds to carry out this research under the “High impact research and technology leadership project.”

Author information

Authors and Affiliations

  1. Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016, India

    Nitu Maity, Ashwani Gautam, Saroj Mishra & Vikram Sahai

  2. Lilly Hall of Biological Sciences, Purdue University - West Lafayette, Indiana, USA

    Ankita Thawani

  3. Biochemical Engineering Department, University College London, London, UK

    Anshul Sharma

Authors
  1. Nitu Maity
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ankita Thawani
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anshul Sharma
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ashwani Gautam
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Saroj Mishra
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Vikram Sahai
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Saroj Mishra.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

ESM 1

(DOCX 26 kb)

ESM 2

(DOCX 80 kb)

ESM 3

(DOCX 2250 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Maity, N., Thawani, A., Sharma, A. et al. Expression and Control of Codon-Optimized Granulocyte Colony-Stimulating Factor in Pichia pastoris . Appl Biochem Biotechnol 178, 159–172 (2016). https://doi.org/10.1007/s12010-015-1865-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-015-1865-y

Keywords

Search

Navigation