Production of recombinant human granulocyte-colony stimulating factor by Pichia pastoris

Human granulocyte-colony stimulating factor (hGCSF) 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 pPIC9 vector. 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 hGCSF production during fermentation were investigated. Cell growth and hG-CSF production were found to be optimal at 28C 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 hGCSF concentration reached 100 g/l and 35 mg/l, respectively. Keywords