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Study On Environmental And Control Conditions For Effective Fusion Protein Expression By Pichia Pastoris

Posted on:2009-03-31Degree:MasterType:Thesis
Country:ChinaCandidate:Y P DuFull Text:PDF
GTID:2120360272456710Subject:Biochemical Engineering
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In this paper, the feed strategy and characteristics of glycerol has been studied by means of DO-Stat control and artificial neural network dynamic pattern recognition (ANNPR) control in Pichia pastoris producing fusion protein of HSA-IL-2. The ANNPR-Ctrl based on the on-line DO/pH measurement was more effective in the cell growth phase. It could increase the mass accumulation efficiently in a short time and put forward the HSA-IL-2 expression time of methanol induction greatly. While in the induction phase, methanol concentration must be controlled at a definited level for effective fusion protein expression.Meanwhile, the ability of using methanol of recombinant strain (KM71, MutS)is weak,and it couldn't use DO and pH to identify methanol of excess or scarcity. So a methanol monitoring and control system was adopted for feedback control of methanol in the induction phase. With the system, the methanol concentration can be controlled at a predetermined level for effective fusion protein expression.DO and methanol concentration had key effects on the expression of HSA-IL-2 for the strain(KM71, MutS). In the research, it was founded that the demand of DO for the strain was not much high, and it could be satisfied with using air and regulating agitation rate. After 3 days of induction,in the mode of normal DO and low methanol concentration (DO 20%-60%,methanol concentration 5-8g/L), the highest expression of HSA-IL-2 was 23mg/L, nearly 2.5 times of that in flask.In the mode of high DO and low methanol concentration (DO 80%-120%,methanol concentration 2-5g/L), the expression of HSA-IL-2 was improved by 18%, up to 27 mg/L. While in the mode of DO oscillation and low methanol concentration (methanol concentration 2-7g/L), HSA-IL-2 expression decreased by 36.5%, down to 14.6mg/L. When in the mode of normal DO and moderately high methanol concentration (DO 20%-60%, methanol concentration 15-23g/L), the expression of HSA-IL-2 reached 44.6mg/L. In the induction prophase, the over-high methanol concentration could cause poisoning to cell, unbenefitting to the expression of HSA-IL-2. In addition, limited co-feeding of glycerol in induction phase could effectively improve cell respiratory activity and promote cell growth to relieve the toxicity caused by methanol.The recombinant strain(GS115, Mut+)expressing HSA-CP was studied in this paper too. It was founded that the demand of DO for the strain was much high. The maximum cell concentration of OD600 reached 600 with oxygen-enriched air (50%,V/V), about 1.8 times of that with air. The induction performances of this strain were different from the strain(KM71, MutS) which expressed HSA-IL-2.The ability of utilizing methanol for this strain was strongger, and the time of induction was shorter, within 15h of induction, the expression of HSA-CP reached the maximum; while the expression of HSA-CP in flask would reach the maximum after 3 days of induction. The maximum expression of HSA-CP with air was 108mg/L, which was slightly lower than that in flask; while the maximum expression of HSA-CP doubled with oxygen-enriched air, up to 237mg/L.
Keywords/Search Tags:Pichia pastoris, Fusion protein, DO, Methanol concentration
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