Studies On The Fermentative Production Of Recombinant Human Interleukin 11(rhIL-11) In Pichia Pastoris

This article firstly optimized the fermentation conditions for the expression of recombinant human interleukin 11 in Pichia pastoris and the prevention of degradation and polymerization of the expression product through culture flask experiment. Further large scale fed-batch fermentation was developed under the optimized conditions and the fermentation product was identified by SDS-PAGE.The experimental results are as follows:1. The optimal culture and induction conditions were as follows:10% inoculation rate, the best bacteria density for induced protein expression was OD6oo=120-150, the induction time was 70 hours,0.5% methanol, pH value was changed from 6.0 for culture to 6.8, and the fermentation temperature was changed from 30℃ for culture to 25℃, and the temperature was further decerase to 22℃ after 48 hours of induction.2. The degradation and polymerization of target protein was significantly reduced. The recombinant Pichia pastoris was further fed with B Braun D100 150L fermentation tank. The optimized fermentation conditions are:Glycerol culture stage:Glycerol concentration was 40g/L, culture temperature 30℃, pH6.0, DO 40%.Glycerol feeding stages:After the first stage (dissolved oxygen increased dramatically),50% glycerol (containing 12ml/L PTM1) at a rate of 24mL/L/h were added, about 4 hours later, OD600 probably reached 120-150, stop feeding.Methanol feeding stage:At the end of the second stage, keep the yeast cell hungry for half an hour or so. When the dissolved oxygen and pH were significantly increased,0.5% methnol was added. At the beginning of the induction, the 1% yeast extracts,2% peptone,0.3% casein, 5mM EDTA,0.1M arginine and 0.1% Twain 20 were added, the temperature was controlled at 25℃, pH6.8.0.3% casein and 0.1% Twain 20 were added every 24h. The temperature was reduced to 22℃ after 48 hours of induction. The total induction time was about 70h. Finally, the protein expression level is more than 1.2g/L in supernanent. The whole processes of the fermentation consist of three stages, about 95h.3. The expression products were analyzed by SDS-PAGE gel electrophoresis. The results showed that the size of expressed protein has the same molecular weight with the rhIL-11. The recombinant human interleukin 11 expression levels has been relatively stable, probably around 1.2g/L, monomer content greater than 0.7g/L at the optimized conditions.