【摘要】 本文对重组人白细胞介素4高效表达克隆pBV220/hIL-4a的表达产物进行了纯化,升对纯化的人IL-4进行了N端氨基酸序列分析。人IL-4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、复性浓缩、离子交換和凝胶过滤层析一系列纯化步骤,终产物纯度达98％以上,按蛋白总量计算回收率为14％,比活性达2×10~6单位/mg蛋白。通过测定纯化人IL-4的N端16个氮基酸序列,与由其DNA序列推导的氨基酸序列完全一致。本文为重组人IL-4的批量生产奠定了基础。更多还原

【Abstract】 Recombinant human interleukin 4 was expressed as inclusion bodies in E. coli. A simple and effective protocol has been worked out for the purification. The hIL-4 was purified to more than 98% homogeneity, the total protein recovery was 14％ and specific activity of hIL-4 was 2×10~6U/mg. There are two key points in our protocol: (1) the improvement of the extraction of inclusion bodies which leads to the hIL-4 comprising up to 80％ of crude extract.(2) during the refolding process, Gdn·HC1 dissolved hIL-4 was diluted into large volume of renaturation buffer to avoid precipitation. The sequence of N-terminal 16 amino acid residues of purified hIL-4 was determined and found to be identical to the native protein.更多还原