六聚组氨酸在重组蛋白中的位置对镍亲和层析的影响

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。

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

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.