High-density Expression of Recombinant Porcine Insulin Precursor by Pichia pastoris

Abstract:

The recombinaut porcine insulin precursor(PIP)produced by Pichia pastoris in shake-flask and 501.fermenter was investigated respectively.The results indicated that 60h induction time length and 2.0%～2.5% methanol addition every day was optimum in shake- flask.The process in 50L fermenter was consisted of batch,feed-batch and induction phases.The relationship between dry cell weight(y) and culture time (t) in growth phase(batch and feed-batch phase)could be described by model y=0.6525e^0.1907t.Glycerol and ammonia were almost used for cell growth and maintain,and no by-product was observed in batch and fed-batch phase.Only 80% ammonia and 70% methanol were used by cell in induction phase. By comparison the results of shake-flask and 50L fermenter,it was concluded that the limit-ing factor in the fermentation of shake-flask and 50L fermenter was dissolved oxygen (DO) and carbon source,respectively. When scaling the result of shake-flask to 50L fermenter,the control strategy was adqpted for 50L fermenter by increasing the feed rate of methanol and the maximum PIP concentration reached 1.72g/L.

Key words:Pichia pastoris; High-density fermentation; Recombinant porcine insulin precursor