Abstract
BackgroundRecombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is a glycoprotein that has been approved by the FDA for the treatment of neutropenia and leukemia in combination with chemotherapies. Recombinant hGM-CSF is produced industrially using the baker’s yeast, Saccharomyces cerevisiae, by large-scale fermentation. The methylotrophic yeast, Pichia pastoris, has emerged as an alternative host cell system due to its shorter and less immunogenic glycosylation pattern together with higher cell density growth and higher secreted protein yield than S. cerevisiae. In this study, we compared the pipeline from gene to recombinant protein in these two yeasts.ResultsCodon optimization in silico for both yeast species showed no difference in frequent codon usage. However, rhGM-CSF expressed from S. cerevisiae BY4742 showed a significant discrepancy in molecular weight from those of P. pastoris X33. Analysis showed purified rhGM-CSF species with molecular weights ranging from 30 to more than 60 kDa. Fed-batch fermentation over 72 h showed that rhGM-CSF was more highly secreted from P. pastoris than S. cerevisiae (285 and 64 mg total secreted protein/L, respectively). Ion exchange chromatography gave higher purity and recovery than hydrophobic interaction chromatography. Purified rhGM-CSF from P. pastoris was 327 times more potent than rhGM-CSF from S. cerevisiae in terms of proliferative stimulating capacity on the hGM-CSF-dependent cell line, TF-1.ConclusionOur data support a view that the methylotrophic yeast P. pastoris is an effective recombinant host for heterologous rhGM-CSF production.
Highlights
Recombinant human granulocyte-macrophage colony-stimulating factor is a glycoprotein that has been approved by the Food and Drug Administration (FDA) for the treatment of neutropenia and leukemia in combination with chemothera‐ pies
It induces an immune reaction because N-formyl methionine is its first amino acid. rhGM-CSF produced in mammalian hosts has a similar glycosylation pattern to the native human protein, but production rates are slow [6, 7]
We show that P. pastoris secretes a higher yield of more active recombinant rhGM-CSF than S. cerevisiae
Summary
Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is a glycoprotein that has been approved by the FDA for the treatment of neutropenia and leukemia in combination with chemothera‐ pies. RhGM-CSF produced in Escherichia coli is biologically active but unstable in human plasma. It induces an immune reaction because N-formyl methionine (fMet) is its first amino acid. RhGM-CSF produced in mammalian hosts has a similar glycosylation pattern to the native human protein, but production rates are slow [6, 7]. The product from baker’s yeast, Saccharomyces cerevisiae, is glycosylated and approved by the FDA for the treatment of neutropenia and leukemia in combination with chemo- or radio-therapy for cancer or transplantation patients. The drawbacks of using S. cerevisiae are hyper-glycosylated products and low cell density growth
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