Ben Ghanem A, Winchenne J J, Lopez C, Chrétien S, Dubarry M, Craescu C T, Le Caer J P, Casadevall N, Rouger P, Cartron J P
Institut National de Transfusion Sanguine, Paris, France.
Prep Biochem. 1994 May;24(2):127-42. doi: 10.1080/10826069408010087.
A recombinant human erythropoietin (rH-EPO) was obtained from the culture supernatants of human B-lymphoblastoid cells transfected by the human EPO gene. rH-EPO was purified by a two-step method based on immunoaffinity and ion exchange chromatography. The first step was achieved by an anti-EPO monoclonal antibody (Mab). This Mab, immobilized on Sepharose 4B, allowed a 410-fold purification of the protein. The second step consisted of ion exchange chromatography on DEAE Sephacel. The combination of these two steps results in a highly purified rH-EPO with a global yield of about 50%; the specific activity of the protein was 176,000 IU/A280. The NMR spectrum was characteristic for a well structured, single-conformation protein. The purified protein was analyzed by SDS-PAGE and isoelectric focusing. The biological activity of purified rH-EPO was measured in vivo, by the incorporation of 59Fe into red blood cells (RBC) of polycythemic mice and in vitro by the proliferative response of an EPO-dependent cell line. The purified protein expressed in lymphoblastoid cells of human origin had the same biological activity as that of urinary EPO and rH-EPO produced in other mammalian cells.
重组人促红细胞生成素（rH-EPO）是从转染了人促红细胞生成素基因的人B淋巴母细胞的培养上清液中获得的。rH-EPO通过基于免疫亲和和离子交换色谱的两步法进行纯化。第一步通过抗EPO单克隆抗体（Mab）实现。这种固定在琼脂糖4B上的单克隆抗体可使蛋白质纯化410倍。第二步包括在DEAE Sephacel上进行离子交换色谱。这两步结合可得到高度纯化的rH-EPO，总产率约为50%；该蛋白质的比活性为176,000 IU/A280。核磁共振谱是结构良好的单构象蛋白质的特征。纯化后的蛋白质通过SDS-PAGE和等电聚焦进行分析。纯化的rH-EPO的生物活性通过体内将59Fe掺入多血质小鼠的红细胞（RBC）以及体外通过EPO依赖性细胞系的增殖反应来测定。在人源淋巴母细胞中表达的纯化蛋白质具有与尿促红细胞生成素以及在其他哺乳动物细胞中产生的rH-EPO相同的生物活性。
