用三步法纯化重组人促红细胞生成素

Purification of recombinant human erythropoietin to homogeneity by a rapid three-step procedure

在生物反应器中培养中国仓鼠卵巢细胞—促红细胞生成素(CHO-EPO)C2细胞株,培养上清液中重组人促红细胞生成素(rHuEPO)表达水平达2000～3000U/ml。培养上清经过三步纯化:第一步为反相柱色谱,可将样品浓缩约96.7％,其收集液经充分透析后进行第二步的DEAE-离子交换色谱,最后进行分子筛色谱,总回收率为30％以上,经纯化的rHuEPO比活性为1.5×10~8U/g蛋白,SDSPAGE一条带,扫描测试纯度达98％以上。

CHO-EPO C2 cells were cultured in the Packed Bed Bioreactor with free-FBS medium. Conditioned medium from bioreactor containing 2000-3000U/ml of recombinant Human erythropietin(rHuEPO). Crude rHuE-PO from conditioned medium was purified using three-step procedure to yield preparation with potencies of 1. 5× 108U/g in 30% yield. The three steps includ: (1) reverse-phase chromatography which made 30-fold concentration, (2) sufficiently dilyzed and purified with ion-exchange chromatography, (3) gel filtration. SDS-PAGE result of the final products revealed a single band, which purity was over 98% with measurment of UV scanning.