STUDIES ON CONDITIONS OF CELL HIGH DENSITY CULTIVATION FOR GENETICALLY ENGINEERED METHYLOTROPHIC PICHIA PASTORIS / 微生物学通报

ABSTRACT

Recently the methylotrophic Pichia pastoris,with many advantages such as high expression,stable genetics and protein secretion,has been used extensively as a host expressing heterologous proteins.The optimal seed medium among seven media MM,MD,MGY,BMGY,YMPD,YMPGy and MGyB is BMGY with addition of 4mL/L PTM1 The high density synthesized medium in shake flask culture is as follows: Glycerol 4%(NH 4) 2SO 4 10g/L,CaSO 4 0.93g/L,K 2SO 4 18 2g/L,MgSO 4?7H 2O 14 9g/L,add 0.1mol/L PBS(pH=6 0) to 1 0 liter.The harvested yeast cell optical density (OD 600 ) reached 65 or more after 26 hours cultivation.By analysis of SDS\|PAGE,the results of Pichia pastoris culture by methanol inducement for 72 hours showed that the expression of recombinant human serum albumin had been achieved by methanol inducement after 12 hours,and the mount of targeted protein reached the summit after 24 hours inducement by methanol.The high density synthesized medium in shake flask culture in this experiment,which is similar to the mediums of batch fermentation and batch\|fed fermentation,is benefit to direct the P.pastoris fermentation in the fermentor.