摘要
表达人IL-2的E.coli pETI-2/MM294在色氨酸饥饿的培养基中发酵,加入一定量的3—吲哚乙酸诱导其trp启动子,可以使rlL-2的表达量高达总菌体蛋白量的19％。高水平表达的rlL-2以不溶性的包含体形式存在于E.coli的胞浆中。通过破碎细菌、变性抽提和使rlL-2复性后,再经CM-SephadexC-50、DEAE-SephadexA-50、SephadexG-75等一系列层析步骤纯化,最终得到的rlL-2纯度达90％以上,比活性为7.21×10~5μ/mg,总回收率为10.5％。
E.coli pETI-2/MM294, a recombinant organism that harbours a plasmid containing human IL- 2 sequence and the trp promoter, was cultured. Recombinant IL- 2 (rIL- 2) expression was induced by the addition of inducer, iadolyl - 3-acrylic acid in the trptophan starvation medium. On this condition, rIL- 2 was expressed at the high level of 19% of total cellular protein as insoluble inclusion bodies in cytoplasm. After cell breakage, rIL- 2 aggregates were isolated and then dissolved in 7M guanidine hydrochloride. Renaturation was effected by dilution and oxidation in the present of GSSG/GSH. Final purification was performed by successive chromatograph steps including CM-Sephadex C-50, DEAE-Sephadex A-50 and Sephadex G-75, yielding over 90% pure rIL-2 with the activity of 7.21×106u/mg and overall recovery of 10.5%.
出处
《免疫学杂志》
CAS
CSCD
北大核心
1990年第4期229-234,共6页
Immunological Journal
关键词
大肠杆菌
白细胞介素
重组
Recombinant IL- 2 , Escherichia coli, High-level expression, Purification
