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

研究了毕赤酵母Pichia pastoris表达的重组人复合α干扰素(cIFN)时不同诱导甲醇浓度对cIFN分离纯化得率的影响,并分析了原因.在5L罐中采用0.25、0.50和0.75％(W/V)三个甲醇浓度诱导时,在0.75％高甲醇浓度诱导下cIFN表达水平最高,达到2.06 g/L,是0.25％低甲醇浓度诱导的1.24倍,但是低甲醇浓度诱导下cIFN分离纯化得率却高于高甲醇诱导浓度下3.75倍.另外,低甲醇浓度下发酵上清液cIFN抗病毒活性为2.85×108IU/mL,较高甲醇浓度提高了4.48倍.进一步采用SDS-PAGE和Native-PAGE免疫印迹分析不同条件下发酵液中cIFN存在状态,发现在高甲醇浓度下cIFN容易形成大量的聚合体,分别为共价聚合和非共价聚合,而cIFN单体含量较少,但是低甲醇浓度诱导下情况完全相反.最终在0.25％甲醇诱导下分离纯化1L发酵上清液可得0.73 g单体cIFN,是0.75％甲醇诱导下的3.84倍.

Influence of methanol concentration on purification recovery of consensus interferon-a produced by Pichia pastoris

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.