His融合蛋白在大肠杆菌中的表达_纯化和EMSA实验.pdfVIP

ISSN 1007-7626 20038 CN 11-3870Q Chinese Journal of Biochemistry and olecular Biology 19(4):457~ 462 Lys -PAI-1 10 * 孙兴会, 李 平, 张宇清, 谭 理, 王 霞, 侯 敏, 陈怡韵, 朱运松 ( 复旦大学上海医学院分子遗传教研室, 上海 200032) 用PCR 方法构建10 个赖氨酸与纤溶酶原激活 抑制物-1的融合基因Lys10-PAI-1,并克隆于 pET28a(+ )和pET32a(+ )原核表达载体. 将重组表达质粒pET32-PAI 和pET28-PAI 转化大肠杆菌 BL21(DE3). IPTG诱导后可获得分子量分别为63 kD 和43kD 的目的蛋白, 表达蛋白占菌体蛋白 20%以上,大多数重组蛋白以不溶形式存在. 表达产物经变性复性超滤透析和亲和层析等步 骤,可以得到纯化的TrxPAI-1和rPAI-1重组蛋白. Western 印迹结果表明,目的蛋白具有人PAI-1 的抗原性. 凝胶阻滞实验发现,纯化的重组蛋白在 一定条件下, 可以与质粒结合, 使质粒的迁移率 明显改变. 研究结果表明,TrxPAI-1和rPAI-1有希望成为受体介导基因转移的配体. 纤溶酶原激活 抑制物-1,凝胶阻滞实验,融合基因,表达与纯化 Q78 Expression, Purification of Lys -PAI-1 in E . coli and 10 Its Gel Retardation Experiments * SUN Xing-hui,LIPing, ZHANG Yu-qing,TAN Li,WANG Xia, HOU min,CHEN Y-i yun,ZHU Yun-song ( Department of Molecular Genetics, Medical School of Fudan University , Shanghai 200032, China) Abstract The fused gene (Lys -PAI-1) of ten lysine and plasminogen activator inhibitor-1was cloned into 10 prokaryotic expression vector pET32a(+ ) andpET28a(+ ). The recombinant expressionplasmidpET32-PAI and pET28-PAI were transformed into E. coli BL21( DE3). After induction for 3 hours by IPTG, the expressionrecombinants showed a high level expression of recombinant proteinwith bothTrxPAI-1and rPAI- 1 being more than 20% of the total bacterial protein. SDSandWestern blot analysis indicatedthat t