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

ABSTRACT

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