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

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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https://hal-riip.archives-ouvertes.fr/pasteur-00743157
Déposant : Institut Pasteur Tunis <>
Soumis le : jeudi 18 octobre 2012 - 11:49:12
Dernière modification le : vendredi 29 mai 2020 - 19:38:12
Document(s) archivé(s) le : samedi 17 décembre 2016 - 02:12:52

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Najla Gasmi, Rabeb Lassoued, Atef Ayed, Brigitte Tréton, Didier Chevret, et al.. Production and characterization of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) expressed in the oleaginous yeast Yarrowia lipolytica.. Applied Microbiology and Biotechnology, Springer Verlag, 2012, 96 (1), pp.89-101. ⟨10.1007/s00253-012-4141-x⟩. ⟨pasteur-00743157⟩

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