大肠杆菌表达的重组人粒细胞-巨噬细胞集落刺激因子的纯化  

Purification of Recombinant Human Granulocyte-macrophage Colony Stimulating Factor Expressed in Escherichia coli

作　　者：凌明圣[1] 邹民吉[1] 徐明波[1] 王嘉玺[1] 马贤凯[1] 

机构地区：[1]军事医学科学院基础医学研究所,北京100850

出　　处：《生物工程学报》1995年第3期217-221,共5页Chinese Journal of Biotechnology

基　　金：总后"八五"攻关项目

摘　　要：对重组人粒细胞－巨噬细胞集落刺激因子（ｒｈＧＭ－ＣＳＦ）高效表达克隆ｐＺＷ．ＧＭ的表达产物进行了纯化，并对纯化的ＧＭ－ＣＳＦ进行了Ｎ端氨基酸序列分析。人ＧＭ－ＣＳＦ基因表达产物在大肠杆菌中以不溶性包涵体形式存在，经过超声破菌、包涵体抽提、凝胶过滤层析、复性、离子交换一系列化步骤，终产物纯度达９９％，按蛋白总量计算回收率达１０％，比活性达１×１０＾７ｕ／ｍｇ蛋白质。Recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) was expressed as inclusion bodies (IB) in E. coll. A simple and effective protocol has been worked out for the purification. IB collected after the breakage of bacteria through sonication were subjected to repeated washing, and then solubilized in TE buffer (50mmol/L Tris-HCl, Immol/L EDTA, pH8.3) contained 8mol/L urea and 10mmol/L DL-dithiothreitol. By means of Sephacryl-200 HR, refolding and Q Sepharose Fast Flow, rhGM-CSF was obtained with a purity of 99%. The total protein recovery was 10% and specific activity of rhGM-CSF was 1X107u/mg. The sequence of N-termi-nal 16 amino acid residues of purified rhGM-CSF was determined and found to be identical to the native protein. This study provided useful parameters for mass production of rhGM-CSF.

关 键 词：重组人粒细胞 巨噬细胞 集落刺激因子 RHGM-CSF 

分 类 号：R392－33[医药卫生—免疫学]