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Prevention of human granulocyte colony‐stimulating factor protein aggregation in recombinant Pichia pastoris fed‐batch fermentation using additives
Author(s) -
Bahrami Ali,
Shojaosadati Seyed Abbas,
Khalilzadeh Rasoul,
Mohammadian Jafar,
Farahani Ebrahim Vashghani,
Masoumian Mohammad Reza
Publication year - 2009
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20070267
Subject(s) - pichia pastoris , alcohol oxidase , recombinant dna , fermentation , glycerol , yeast , methanol , pichia , granulocyte , chemistry , biochemistry , granulocyte colony stimulating factor , biology , gene , immunology , organic chemistry , genetics , chemotherapy
rhG‐CSF (recombinant human granulocyte colony‐stimulating factor) was expressed in the yeast Pichia pastoris under the control of the AOX1 (alcohol oxidase 1) promoter. The production of rhG‐CSF was induced by switching from growth on glycerol to growth on methanol. In the induction phase, the methanol feed rate had a significant effect on the specific expression rate of rhG‐CSF. A constant feed rate of 16 ml·h −1 ·l −1 was found to be optimal for a high specific expression rate of rhG‐CSF (0.058 mg −1 ·h −1 ·g DCW −1 ; DCW is dry cell weight). Under this condition, a maximum concentration of 300 mg/l of rhG‐CSF and the expression yield of 0.6 mg of rhG‐CSF/g of methanol were attained. However, the secreted rhG‐CSF was shown to exist as aggregates in the culture broth, owing to hydrophobic interactions. To prevent undesirable protein aggregation, the presence of additional additives in the P. pastoris culture medium was investigated. Among seven additives tested, Tween 20, Tween 80 and betaine exhibited the best results in respect of preventing the formation of rhG‐CSF protein aggregates.