Prevention of human granulocyte colony-stimulating factor protein aggregation in recombinant Pichia pastoris fed-batch fermentation using additives.
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.
Bahrami
Ali
A
Biotechnology Group, Department of Chemical Engineering, Faculty of Engineering, Tarbiat Modares University, PO Box 14155-143, Tehran, Islamic Republic of Iran.
Shojaosadati
Seyed Abbas
SA
Khalilzadeh
Rasoul
R
Mohammadian
Jafar
J
Farahani
Ebrahim Vashghani
EV
Masoumian
Mohammad Reza
MR
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000494:pharmacology; D001483:Base Sequence; D001622:Betaine / Q000494:pharmacology; D049109:Cell Proliferation; D003470:Culture Media; D005285:Fermentation; D016179:Granulocyte Colony-Stimulating Factor / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D000432:Methanol / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia / Q000166:cytology / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins; D013501:Surface-Active Agents / Q000494:pharmacology
D000596:Amino Acids; D003470:Culture Media; D011994:Recombinant Proteins; D013501:Surface-Active Agents; D016179:Granulocyte Colony-Stimulating Factor; D001622:Betaine; D011092:Polyethylene Glycols; D000432:Methanol