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

作　　者：吴丹[1] 储炬[2] 王永红[2] 庄英萍[2] 张嗣良[2] 

机构地区：[1]江南大学食品科学与技术国家重点实验室,无锡214122 [2]华东理工大学生物反应器国家重点实验室,上海200237

出　　处：《生物工程学报》2011年第12期1789-1796,共8页Chinese Journal of Biotechnology

基　　金：国家重点基础研究发展计划(973计划)(No.2007CB714306);生物反应器国家重点实验室特别基金(No.2060204)资助~~

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。We observed the influence of methanol concentration on purification recovery of recombinant human consensus interferon-a （clFN） produced by Pichiapastoris. Fermentations controlled at 0.75% （W/V） methanol showed a 24% increase in clFN expression compared to using 0.25% methanol. However, the purification recovery rate of clFN at 0.25% methanol was 3.75-fold higher than that at 0.75% methanol. To seek the reason, we analyzed the stability of clFN by SDS-PAGE and Native-PAGE as well as Western blotting. The electrophoresis results revealed that clFN formed a lot of aggregates in media when the induction wascontrolled 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 supematant 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.

关 键 词：毕赤酵母 复合α干扰素 甲醇浓度 聚合体 

分 类 号：Q78[生物学—分子生物学]