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.