Influence of methanol concentration on purification recovery of consensus interferon-a produced by Pichia pastoris
甲醇浓度对毕赤酵母表达重组人复合α干扰素分离纯化得率的影响

We observed the influence of methanol concentration on purification yield rate of recombinant human consensus interferon (cIFN) produced by Pichia pastoris. Fermentations controlled at 0.75% (W/V) methanol showed a 24% increase in cIFN expression compared to the induced culture at 0.25% methanol. However, a sharp drop of 79% in purification yield rate of cIFN was seen at 0.75% methanol compared to 0.25% methanol. To seek the reason, we analyzed the stability of cIFN by SDS-PAGE and Native-PAGE as well as Western blotting. The electrophoresis results revealed that cIFN formed a lot of aggregates in media when the induction was controlled at 0.75% methanol, and two different aggregate forms were found: disulfide bond covalent aggregates and non-covalent aggregates. However, these aggregates almost disappeared when the methanol concentration was controlled at 0.25%, at the same time, cIFN bioactivity of supernatant increased almost 4.48-fold. It is the first time to report the influence of methanol concentration on stability of recombinant protein by P. pastoris. Our results strongly emphasizes the necessity to obtain a maximum of information on the process parameters such as methanol concentration and to maintain strict process control in order to increase the quality of recombinant protein in the high cell-density fermentation of P. pastoris.