重组人载脂蛋白AⅠ米兰突变体在酵母中的分泌表达

Secretion Expression of Human Recombinant ApoAⅠ-milano in Yeast Pichia pastoris

将重组质粒pPIC9K-apoAⅠ-milano用BglⅡ酶切后转化Pichia pastorisGS115菌株,筛选得到G418高抗性转化子,经甲醇诱导和摇瓶发酵,用SDS-PAGE与Western blot检测上清液,发现有明显的重组人载脂蛋白AⅠ米兰突变体(rAIM)表达,但其分子量比正常蛋白小.对酵母工程菌的摇瓶发酵条件进行优化后,rAIM表达水平得到提高,约为70 mg/L发酵液,其中正常大小的rAIM所占的比例约为80%.采用冷丙酮沉淀法初步纯化了酵母工程菌发酵液中的rAIM,并验证其有磷脂结合能力.

The recombined pPlC9K-apoA Ⅰ-milano was digested by Bgl Ⅱ and transformed into Pichia pastoris strain GS115, then the transformants with high G418 resistance were selected. After the transformants growing in the shaking flask and were induced by methynol, the SDS-PAGE and Western blot analysis of supernatant showed that they could express rAIM in a high level, but the C terminal is truncated. Shake cultivation of the strain using the optimized condition yielded 70 mg rAIM per liter of supematant, the full length protein reached 80 % of total rAIM. The cold acetone sedimentation method was used to partly purify the rAIM in the fermentation supernatant. Furthermore, the ability of rAIM to combine with lipid was assayed.