title: 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.
authors.affiliation: INRA, UMR1319 Micalis, Domaine de Vilvert, 78350 Jouy-en-Josas, France.
authors.forename: Najla
authors.initials: N
authors.lastname: Gasmi
authors.forename: Rabeb
authors.initials: R
authors.lastname: Lassoued
authors.forename: Atef
authors.initials: A
authors.lastname: Ayed
authors.forename: Brigitte
authors.initials: B
authors.lastname: Tréton
authors.forename: Didier
authors.initials: D
authors.lastname: Chevret
authors.forename: Jean Marc
authors.initials: JM
authors.lastname: Nicaud
authors.forename: Héla
authors.initials: H
authors.lastname: Kallel
mesh_terms: D001709:Biotechnology / Q000379:methods; D015870:Gene Expression; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008970:Molecular Weight; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D025062:Yarrowia / Q000235:genetics*
chemical_list: D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor