Effective PINF-α Expression In Pichia Pastoris By Multi-variables On-line Control

In this study, recombinant pichia psatoris KM71H(IFNα-pPICZαA) was chosen as a experimental strain to express porcine interferon-α(pIFN-α) by high cell density culture conducted in a 5L and 10L fermentor. The key variables that influenced pIFN-αantiviral activity were analyzed systematically by many experimental runs contrast. The main results were described as follows:(1) Changing patterns of OUR is a monitoring indicator which determine the methanol concentration level by researching various variables such as OUR,CER and RQ during induction phase. High and stable OUR should be obtained during growth phase for effective pIFN-αexpression production. It could be obtained by controlling the methanol at a level of 10g/L and controlling specific growth rate during mixed phase of glycerol and methanol. The highest pIFN-αantiviral activity could reach a level of 6.6×10~6 IU/ml when setting a moderate methanol concentration level, which was more than 10~300 folds higher than those obtained at high methanol and low methanol concentration in using same fermentor.(2) Autocontroling the rate of feeding substrate at a moderate level during transition phase,it could reach high and glycerol consumption rates by many experiments using 5 L fermentor. Before induction phase (fermentation at 32h), it could reach high cells density about 100g/L above. At this condition, OUR and RQ could be controlled a level of 250mmol/L/h and 0.4. The highest pIFN-αantiviral activity could reach a level of 1×10~6 IU/ml above.(3) Cell growth rate, glycerol consumption rates,OUR of 10L fermentor was slower than that of 5L fermentor by the same controlling condition. The total protein content(the target protein is about 70% of that ) is only 1/5~1/4 of that 5 L fermentor. High and stable pIFN-αantiviral activity should be increased in the following experiments.