A Plackett-Burman design was used to evaluate the factors related to rmHSA-GCSF expression by pichiapastoris in shake flask. Induction pH, methanol concentration and induction time with significant influence on rmHSA-GCSF fermentation were selected. The response surface methodology was used to obtain the optimal region of the three factors. The optimal process conditions were induction pH 5.54, methanol concentration 2.45% and induction time 66.4 h. Under
these conditions, the maximum predicted yield of rmHSA-GCSF was 157.5mg/L. According to the optimization of culture conditions in shake flask, the rmHSA -GCSF yield was increased up to about 600 mg/L in 150 L fermentor. High -level expression of rmHSA -GCSF was successfully achieved.