pH对毕赤酵母表达重组人复合a干扰素的降解影响

Effect of pH on Proteolytic Degradation of Consensus Interferon-α Expressed by Pichia pastoris

摘要:

使用Pichia pastoris表达重组人复合a干扰素(cIFN)会发生降解、聚合等不均一表达的现象。在5 L发酵罐中考察了不同诱导pH对cIFN表达产生降解的影响, 结果发现在适合酵母生长的pH 3.0~7.0范围内, 当诱导pH为4.0~5.0时, cIFN不均一表达现象最少, 生物活性达到2.5×108 IU/mL。通过测定发酵液中总蛋白酶活和细胞活性寻找了cIFN降解出现的原因：发现低诱导pH下细胞死亡率升高释放更多酶系, 高诱导pH下蛋白酶活性明显增大, 两者都使蛋白酶作用加强, 加剧cIFN的降解; 特别是诱导pH为7.0时, 适宜的pH使蛋白酶酶活陡升, 将cIFN完全降解。

Abstract:

We observed the heterogeneity of consensus interferon (cIFN) expressed by Pichia Pastoris, such as aggregation and degradation. The effect of induction pH on degradation of cIFN was studied in a 5-L bioreactor. The heterogeneity of cIFN was less serious when induced at pH 4.0~5.0. The bioactivity of the fermentation supernatant reached 2.5×108 IU/mL when pH was controlled between 4.0 and 5.0. To seek the reason of cIFN degradation, we analyzed the activity of protease and cell mortality by flow cytometry. At lower pH, cell mortality was higher and more protease was released. Protease activity increased at higher pH. The proteolytic degradation of cIFN was enhanced at either high or low pH. The cIFN was degraded entirely when pH was 7.0, where the protease had its optimal pH.