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

用生物反应器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２×１０６～３×１０６Ｕ／Ｌ。培养上清液经过三步纯化：第一步为反相色谱，可将样品体积浓缩约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１．５×１０８Ｕ／ｇ蛋白，ＳＤＳＰＡＧＥ为一条带，扫描测试纯度达９８％以上。

Chinese hamster ovary erythropoietin (CHO EPO) C2 cells were cultured in the packed bed bioreactor with free fetal bovine serum medium. Conditioned medium from the Bioreactor contained 2×10 6-3×10 6 U/L of recombinant human erythropoietin (rHuEPO). Crude rHuEPO from the conditioned medium was purified by three step procedure to yield preparations with potency of 1.5×10 8 U/g in 30% yield. The three steps involved: (1)reverse phase chromatography which made 30 fold concentration; (2)sufficient dialysis and purification with ion exchange chromatography; (3)gel filtration. SDS PAGE result of the preparations showed a single band. Homogeneity was confirmed by UV scanning.