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A Novel Methanol-Free Platform for Extracellular Expression of rhGM-CSF in Pichia pastoris

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Abstract

The production of the recombinant proteins under the control of AOX1 promoter is a one of the most common expression systems in the methylotrophic yeast Pichia pastoris which is induced by methanol. The application of this expression platform is restricted by the toxicity and inflammatory nature of methanol, especially in food and pharmaceutical products. Human granulocyte macrophage-colony stimulating factor (hGM-CSF) is an important pharmaceutical protein, playing a crucial role in the proliferation and differentiation of innate immune cells. In this study, a methanol-free expression platform for extracellular expression of hGM-CSF was developed. To attain this goal, a novel constructed expression vector pEP(α)101, carrying the FMD promoter regulating recombinant expression by glycerol derepression was designed. The optimized hGM-CSF gene was subcloned into pEP(α)101 and transformed into P. pastoris. The expression of rhGM-CSF in three different culture media were investigated. Based on the observed heterogeneous glycosylation pattern on SDS-PAGE and western blot, the glycoproteins were deglycosylated to remove carbohydrate units. According to the results, the novel methanol independent PFMD expression platform would be a suitable candidate for driving heterologous gene expression especially for the production of food-grade and therapeutically important recombinant proteins.

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Acknowledgements

This work has been financially supported by University of Tehran (28850/06/05).

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  1. Roghayeh Shirvani and Sajjad Yazdanpanah contributed equally to this work.

Authors and Affiliations

  1. Department of Life Science Engineering (LSE), Faculty of New Sciences and Technologies (FNST), University of Tehran, Tehran, Iran

    Roghayeh Shirvani, Sajjad Yazdanpanah & Mohammad Barshan-tashnizi

  2. Department of Quality Control, Research and Production Complex, Pasteur Institute of Iran, Tehran, Iran

    Maryam Shahali

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  1. Roghayeh Shirvani
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Correspondence to Mohammad Barshan-tashnizi or Maryam Shahali.

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Shirvani, R., Yazdanpanah, S., Barshan-tashnizi, M. et al. A Novel Methanol-Free Platform for Extracellular Expression of rhGM-CSF in Pichia pastoris. Mol Biotechnol 61, 521–527 (2019). https://doi.org/10.1007/s12033-019-00182-6

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