重组巴氏毕赤酵母高密度发酵表达rHSA  

Expression of Recombinant Human Serum Albumin in Genetically Engineered Pichia pastoris in High-density Fermentation

作　　者：郭美锦[1] 庄英萍[1] 储炬[1] 张嗣良[1] 刘志敏[2] 

机构地区：[1]华东理工大学生物反应器工程国家重点实验室,上海200237 [2]军事医学科学院生物工程研究所,北京100071

出　　处：《微生物学报》2002年第1期62-68,共7页Acta Microbiologica Sinica

基　　金：国家自然科学基金资助项目 ( 2 9976 0 1 3);教育部高等学校骨干教师资助计划资助项目~~

摘　　要：对基因工程菌Pichiapastoris的摇瓶发酵条件进行了试验 ,并根据摇瓶发酵的优化结果进行了补料分批高密度发酵。在摇瓶发酵时 ,甲醇诱导基因工程菌P .pastoris表达重组人血清白蛋白的发酵周期为 96h ;甲醇的最佳诱导浓度为 1 0g L ;发酵pH范围为 5 72～ 6 5 9;在摇瓶培养时 ,随着接种量的增加 ,虽然目的蛋白表达量缓慢增加 ,但单位细胞光密度的蛋白产率却明显下降 ,符合y =1 2 941x- 0 50 59方程 (线性相关系数r=0 9789) ,其限制性因子很可能为溶氧。在分批发酵 ,接种量为 1 0 %且种子细胞光密度 (OD60 0 )为 2 0左右时 ,细胞生长的延迟期为 2 1 1h左右 ,细胞生长光密度与培养时间的关系模型为 :y =0 7841e0 .2 3 19t(线性相关系数r=0 .993 6 ) ;在补料发酵时细胞干重浓度可达到 1 1 5g L— 1 6 0g L ,在 1 2 0h重组人血清白蛋白表达量最大达到 3 6g L。The optimum culture conditions of genetically engineered Pichia pastoris in shake-flask cultivation and in fed-batch fermentation were investigated respectively in this paper.It showed that the cultural period induced with 5g/L methanol is 96h,optimum methanol concentration is 10g/L,and pH range is 5.72～6.59 in shake-flask culture.Although the seed inoculum amount increased,the target protein productivity per cell optical density was decreased.Their relationship fit the equeation Y 12.941x -0.5059(r 0.9789,where x is inoculating OD 600,Y is protein productivity per cell optical density),we postulated that the restricting factor may be dissolved oxygen (DO) at shake-flask culture.With the 10% inoculum and 20 OD 600 of seed,the lag phase of cell growth is 2 11h in batch cultivation,and the relationship between cell optical density (Y) and culture time (t) is Y 0.7841e 0.2319t(r 0.9936);The cell dry weight of broth reached 115～160g/L and the maximum rHSA concentration was 3.6g/L at the 120 th at the fed-batch fermentation phase.

关 键 词：重组巴斯德毕赤酵母 高密度发酵 重组人血清白蛋白 rHSA 表达 

分 类 号：TQ920[轻工技术与工程—发酵工程]