重组人载脂蛋白A-Ⅰ在毕赤酵母(Pichia pastoris)中的高表达  

Expression of Human Recombinant ApoAⅠin Pichia pastoris

作　　者：冯美卿[1] 蔡钦生[1] 宋大新[2] 钟江[2] 吴满平[1] 周珮[1] 

机构地区：[1]复旦大学药学院生物合成教研室,上海200032 [2]复旦大学生命科学院微生物教研室,上海200433

出　　处：《生物工程学报》2004年第6期906-911,共6页Chinese Journal of Biotechnology

基　　金：海市科委重点项目基金资助 (No .0 2 43 192 0 4)~~

摘　　要：高密度脂蛋白 (High densityLipoprotein ,HDL)是血浆中重要的脂蛋白 ,其主要成分为载脂蛋白AⅠ(ApoliproteinAⅠ ,ApoAⅠ为了大量制备该蛋白 ,首先尝试利用Pichiapastoris表达系统高效表达ApoAⅠ。通过PCR扩增获得天然含人载脂蛋白ApoAⅠ的基因片段 ,将其插入到P .pastoris分泌型载体pPIC9K上 ,BglⅡ酶切线性化后电转化P .pastorisGS115 ,将获得的 10 0 0多个转化子依次在含不同G4 1 8浓度的YPD平板筛选高抗性转化子 ,得到的2 2个高抗性转化子经甲醇诱导 ,SDS PAGE检测得到 6株高表达菌。然后对其中的高表达菌株AP16的培养及诱导条件进行了优化 ,结果显示 :接种后培养 2 4～ 2 8h ,转入诱导阶段 ,培养基pH值在 7～ 7 5 ,菌体密度OD6 0 0 =80左右 ,以 1%甲醇诱导 96h最有利于ApoAⅠ的表达 ,表达水平达 16 0mg L。 14L发酵罐结果显示表达水平与摇瓶相当 ,均高于其它表达系统 。Apolipoprotein AI is an important apoliprotein in human plasma that has a wide range of physiology and pharmaceutical functions. To obtain large quantity of the protein for research and application, Pichia pastoris expression system was first used for high-level expression of recombinant ApoA-Ⅰ. The cDNA fragment encoding human ApoAI was inserted into expression vector pPIC9K and the resulted plasmid was linearized and used to transform P. pastoris GS115 for secretory expression. Twenty-two transformants with high resistance to G 418 were selected from over 1000 transformants. Among the six transformants expressed, recombinant ApoAI was secreted at high level after methanol induction. Western blot analysis indicated that the recombinant ApoAI from P. pastoris was similar to those of native ApoAI from human serum in antigenicity and molecular weight. After growth in BMGY for 28 h, recombinant strain AP16, with cell density of 80 at A 600, was induced for 96h with 1% methanol in BMMY (pH 7.0～7.5) to achieve an expression level of 160 mg/L in both 250 mL shake flask and 14L fermentation, which was much higher than that of other expression systems.

关 键 词：重组人载酯蛋白ApoA Ⅰ 毕赤酵母 表达优化 

分 类 号：Q819[生物学—生物工程]