葡激酶N端突变体的分子设计、高效表达与分离纯化  

Molecular Design, Cloning, Expression and Purification of NH 2 terminal Mutant of Staphylokinase(ΔNSak)

作　　者：宋钢[1] 莫炜[1] 宋后燕[1] 

机构地区：[1]上海医科大学分子遗传学研究室,上海200032

出　　处：《生物化学与生物物理学报》1999年第5期607-609,共3页

基　　金：国家863 计划资助项目

摘　　要：同源模建人微小纤溶酶原（ ｍｉｃｒｏｐｌａｓｍｉｎｏｇｅｎ ， ｍｐｌｇ） 的三维结构， 建立葡激酶（ｓｔａｐｈｙｌｏｋｉｎａｓｅ ， Ｓａｋ） 、ｍｐｌｇ计算机分子对接的结构模型， 模型与已有的实验结果基本相符。为研究葡激酶 Ｎ 端结构与功能的关系， 进一步改造葡激酶分子， 根据复合物结构模型设计了 Ｎ 端缺失１５ 个氨基酸的葡激酶突变体（Δ Ｎ Ｓａｋ） 。Δ Ｎ Ｓａｋ 在大肠杆菌中以可溶性形式表达， 具有纤溶活性。工程菌发酵、压榨后， 经两步离子交换层析分离纯化， 可获纯度达９５ ％ 以上、比活性为９ ．０ ×１０４ Ｉ Ｕ／ｍｇ 的Δ Ｎ Ｓａｋ ， 与野生型 Ｓａｋ 相似。这表明 Ｎ 端１５ 个氨基酸对葡激酶表现纤溶活性影响很小， 并再次验证了复合物结构模型的合理性。The 3 D structure of human microplasminogen was predicted by the method of homology modeling. The binding sites of staphylokinase and microplasminogen were determined by high resolution genetic docking. This model is consistent with several known experimental properties of staphylokinase. To elucidate the function of NH 2 terminus of staphylokinase for further rational reconstruction, a NH 2 terminal deletion mutant of staphylokinase(ΔNSak) was designed . The soluble ΔNSak was achieved with fibrinolytic activity. ΔNSak was purified by two step ion exchange chromatography. The purity was over 95% and the specific activity of ΔNSak was 9×10 4 IU/mg. These data suggest that the 15 NH 2 terminal amino acids of staphylokinase are not required for its plasminogen activating potential and the computer model is reasonable.

关 键 词：葡激酶 分子设计 突变体 表达 纯化 

分 类 号：Q556.3[生物学—生物化学]