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Production and characterization of human granulocyte–macrophage colony-stimulating factor (hGM-CSF) expressed in the oleaginous yeast Yarrowia lipolytica

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Abstract

Since its isolation, the human granulocyte–macrophage colony-stimulating factor (hGM-CSF) has been proposed as a new class of therapeutic biological products in the treatment of various diseases. However, the toxicity of this cytokine towards its expression host constitutes a major obstacle to bioprocess development for large-scale production. In this work, the optimized gene encoding hGM-CSF was expressed in the yeast Yarrowia lipolytica in one and two copies under the control of the fatty acid-inducible POX2 promoter. Protein secretion was directed by the targeting sequence of the extracellular lipase (LIP2): preXALip2. After 48 h of induction, Western blot analysis revealed the presence of a nonglycosylated form of 14.5 kDa and a trail of hGM-CSF hyperglycosylated varying from 23 kDa to more than 60 kDa. The two-copy transformants produced hGM-CSF level which was sevenfold higher compared to the single-copy ones. Deglycosylation with PNGase F showed two forms: a mature form of 14.5 kDa and an unprocessed form of 18 kDa. The addition of two alanines to the signal sequence resulted in correct hGM-CSF processing. The production level was estimated at 250 mg/l after preliminary optimization studies of the cultivation and induction phases. The purified hGM-CSF was identified by N-terminal sequencing and LC-MS/MS analysis; its biological activity was confirmed by stimulating the proliferation of TF1 cell line. This study demonstrated that Y. lipolytica is a promising host for the efficient production of active toxic proteins like hGM-CSF.

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Acknowledgments

The authors would like to acknowledge EGIDE for their financial support in the frame of the CMCU project France–Tunisia (grant no. 07 G0913). LC-MS/MS analyses were performed using PAPPSO (Plateforme d’Analyses Protéomiques Paris Sud Ouest, http://pappso.inra.fr) facilities.

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Authors and Affiliations

  1. INRA, UMR1319 Micalis, Domaine de Vilvert, 78350, Jouy-en-Josas, France

    Najla Gasmi, Brigitte Tréton, Didier Chevret & Jean Marc Nicaud

  2. Unité de Biofermentation, Laboratoire de Microbiologie Moléculaire, Vaccinologie et Développement Biotechnologique, Institut Pasteur Tunis, 13, place Pasteur, BP 74, 1002, Tunis Belvédère, Tunisia

    Najla Gasmi, Rabeb Lassoued, Atef Ayed & Héla Kallel

  3. CNRS, Micalis, 78350, Jouy-en-Josas, France

    Jean Marc Nicaud

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  1. Najla Gasmi
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  2. Rabeb Lassoued
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  3. Atef Ayed
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  4. Brigitte Tréton
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  5. Didier Chevret
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  7. Héla Kallel
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Correspondence to Héla Kallel.

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Gasmi, N., Lassoued, R., Ayed, A. et al. Production and characterization of human granulocyte–macrophage colony-stimulating factor (hGM-CSF) expressed in the oleaginous yeast Yarrowia lipolytica . Appl Microbiol Biotechnol 96, 89–101 (2012). https://doi.org/10.1007/s00253-012-4141-x

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  • DOI: https://doi.org/10.1007/s00253-012-4141-x

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