六聚组氨酸在重组蛋白中的位置对镍亲和层析的影响  

Effects of Position of Hexahistine in Recombinant Protein on Nickel Chelating Affinity Chromatography

作　　者：许艳[1] 万敏[1] 张培因[1] 卫红飞[1] 王丽颖[1] 

机构地区：[1]吉林大学基础医学院分子生物学教研室,长春市130021

出　　处：《医学分子生物学杂志》2004年第2期90-93,共4页Journal of Medical Molecular Biology

摘　　要：目的研究六聚组氨酸在重组蛋白中的位置对镍亲和层析的影响。方法采用PCR法扩增目的基因,克隆人pMD18-T载体后进行序列测定。构建原核表达载体pET28-His-Cpn10-IL4与pET28-Cpn10-His-IL-4,在大肠杆菌菌株BL21(DE3)中表达两种在不同位置带有六聚组氨酸(His-tag)的重组白细胞介素4(rhIL-4)融合蛋白。用SDS-PAGE、Western印迹鉴定表达产物,采用镍亲和层析的方法纯化两种重组融合蛋白。结果Western印迹证实两种融合蛋白均可与抗组氨酸单克隆抗体特异性结合,但用镍亲和层析方法可以很好地分离纯化融合蛋白His-Cpn10-IL-4,却不能纯化融合蛋白Cpn10-His-IL-4。结论应用镍亲和层析的方法纯化融合蛋白时,His-tag在融合蛋白中的位置对蛋白质的纯化非常重要,His-tag可以在融合蛋白的N端和C端,但不能在中间。Objective To investigate the effect of position of hexahistine in recombinant protein on nickel chelating affinity chromatography. Methods Target genes were amplified by PCR, then cloned into pMD18-T and sequenced. Two recombinant expression plasmids-pET28-His-CpnlO-IL-4 and pET28-CpnlO-His-IL-4 were constructed to express two kinds of proteins which have hexahistine (6-His tag) in different positions in E. coli BL21 (DE3 ) respectively. Expression products were identified by SDS-PAGE and Western-blot and purified by Ni2+ affinity chromatography. Results Two kinds of fusion proteins could be combined with anti-histine single cloned antibody. Fusion protein His-Cpn10-IL-4 could be separated and purified by nickel chelating affinity chromatography , while fusion protein His-Cpn10-IL-4 could not be purified by this technique. Conclusion When fusion protein is purified by Ni2+ affinity chromatography, the position of 6-His tag is very important for the purification of fusion protein. 6-His tag could be placed at the amino terminal and carboxy terminal of fusion protein except the middle.

关 键 词：白细胞介素-4 重组蛋白 六聚组氨酸 镍亲合层析 

分 类 号：R341[医药卫生—基础医学]