Production of Recombinant Human Granulocyte-Colony Stimulating Factor by Pichia pastoris

Human granulocyte-colony stimulating factor (hG-CSF) cDNA was expressed in the methylotrophic yeast Pichia pastoris under the control of the alcohol oxidase (AOX1) promoter. An expression vector for hG-CSF secretion was constructed using vector pPIC9. Higher levels of hG-CSF was obtained using a P. pastoris Mut+ (methanol utilization fast) phenotype. The effects of environmental factors such as temperature and pH on the P. pastoris cell growth and hG-CSF production during fermentation were investigated. Cell growth and hG-CSF production were found to be optimal at 28°C and pH 6.0. A fed-batch fermentation process was also developed to obtain high cell density and higher levels of protein expression. Using a high cell density cultivation method, cell dry weight and hG-CSF concentration reached 100 g/l and 35 mg/l, respectively.