Prokaryotic Expression and Purification of Ca~(2+)-ATPase from Aquatic Plant Eichhornia crassipes

Abstract

从水生植物凤眼莲叶片中提取总rnA,经rT-PCr扩增出CA2+-ATPASE基因片段,经限制性内切酶(SMA I,nOT I)酶切后按正确的读码框顺序插入到PgEX-4T-2表达载体上,重组质粒转化大肠杆菌,经菌落PCr和质粒双酶切鉴定、序列测定确认,证实成功地构建了CA2+-ATPASE基因融合表达载体,.转化菌经IPTg诱导表达,获得了大小约48kd的可溶性目的蛋白,与预期相吻合.利用谷胱甘肽琼脂糖凝胶4b(gluTATHIOnE SEPHArOSE 4b)亲和介质对重组蛋白进行纯化,获得了高纯度的目的蛋白.The Ca2+-ATPase gene was cloned fromEichhornia crassipes leaves using the PCR technology.After digested by the enzymes(Sma I,Not I),it was inserted into the plasmid pGEX-4T-2to reconstruct the expression vector.The recombinant protein was induced by IPTG and then purified using Glutathione Sepharose 4B.As a result,a single 48 kDa protein was acquired,which implied the protein was the purified Ca2+-ATPase fusion protein.国家自然科学基金面上项目(30770391