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

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

摘要:

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5 L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2.85×108 IU/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1 L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。

Abstract:

We observed the influence of methanol concentration on purification recovery of recombinant human consensus interferon-α (cIFN) produced by Pichia pastoris. Fermentations controlled at 0.75% (W/V) methanol showed a 24% increase in cIFN expression compared to using 0.25% methanol. However, the purification recovery rate of cIFN at 0.25% methanol was 3.75-fold higher than that at 0.75% 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. Finally, 0.73g monomer cIFN was obtained after purification from 1 liter supernatant at 0.25% methanol induction, showed a 2.84-fold increase compare to the induction at 0.75% methanol.