Process optimization of constitutive human granulocyte-macrophage colony-stimulating factor (hGM-CSF) expression in Pichia pastoris fed-batch culture.
Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is a therapeutically important cytokine that is poorly expressed because of its toxic effects on the host cells. Extracellular expression of hGM-CSF was obtained by cloning its gene in Pichia pastoris under the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter with an N-terminal alpha peptide sequence for its extracellular production. The clones obtained were screened for a hyper producer following which media and cultivation conditions were optimized in shake flasks. Batch and fed-batch studies were performed in a bioreactor where different feed compositions were fed exponentially to obtain high biomass concentrations. Feeding of complex media allowed us to maintain a high specific growth rate of 0.2 h(-1) for the longest time period, and a final biomass of 98 g DCW/l was obtained in 34 h. Product formation was found to be growth associated, and the product yield with respect to biomass (Y (P/X)) was approximately 2.5 mg/g DCW. The above fed-batch strategy allowed us to obtain fairly pure glycosylated hGM-CSF at a final product concentration of 250 mg/l in the culture supernatant with a high volumetric productivity of 7.35 mg l(-1) h(-1).
Centre for Biotechnology, Jawaharlal Nehru University, New Delhi, 110067, India.
Yogender
Y
Pal
Amardeep
A
Khushoo
K J
KJ
Mukherjee
D018533:Biomass; D003470:Culture Media / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D005656:Fungal Proteins / Q000032:analysis / Q000302:isolation & purification; D015966:Gene Expression Regulation, Fungal; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000235:genetics; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D010843:Pichia / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
D003470:Culture Media; D005656:Fungal Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)