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.