[16] Purification of proteins using polyhistidine affinity tags

Abstract

The expression and subsequent purification of recombinant proteins are widely employed in biochemical studies. A powerful purification method involves the use of peptide affinity tags, which are fused to the protein of interest and used to expedite protein purification via affinity chromatography. 1,2 A widely employed method utilizes immobilized metal-affinity chromatography (IMAC) to purify recombinant proteins containing a short affinity tag consisting of polyhistidine residues. IMAC is based on the interactions between a transition metal ion (Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ ) immobilized on a matrix and specific amino acid side chains. Histidine is the amino acid that exhibits the strongest interaction with immobilized metal ion matrices, as electron donor groups on the histidine imidazole ring readily form coordination bonds with the immobilized transition metal. Peptides containing sequences of consecutive histidine residues are efficiently retained on IMAC column matrices. Following washing of the matrix material, peptides containing polyhistidine sequences can be easily eluted by either adjusting the pH of the column buffer or adding free imidazole to the column buffer. 3

Automation of protein purification for structural genomics

“…The affinity tag should be unique, accessible, and preferably small, have high capacity to bind to a matrix and excellent conditional affinity (ON/OFF binding), and should be low cost. The His 6 -tag and its variants appear to meet all of these criteria and are highly effective for protein purification [16]. However, the His 6 -tag-based approach still has a few drawbacks such as, not all proteins can be labeled with His 6 -tag on their N-or C-terminus, the tag may interfere with protein folding or oligomerization, and the tag may be inaccessible or lead to protein aggregation [17].…”

Production and purification of constructed recombinant hirudin in BL21(DE3) strain

“…[19,20] Furthermore, BL21(DE3) strain also sensitive to the ampicillin while pET-16b vector carry ampicillin resistance as selective marker because of that the addition of ampicillin to the medium and the ability of BL21(DE3) strain grown on it due to harboaring pET-16b plasmid. [21] In this study constructive vector pET-16b-HS was extracted (see Figure 4), and by electrophoresis with control the resulted band of constructive vector about 5,901 bp in comparison with control about 5,711 bp that's mean presence of target gene in constructive vector, for more detection from the constructive vector which extracted from transformed cell, Hirudin genes was amplifying with PCR cycle, the resulted band from gel electrophoresis about 202 bp (see Figure 5). To detect gene expression for Hirudin gene, real-time PCR has been done as the results shown in Figures 6&7, threshold curve.…”

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“…Finally, 100 ml of E. coli cell cultures were induced with 0.5 mM IPTG in rich media (Dynamite) and purified by affinity purification using immobilized metal affinity chromatography (IMAC). Affinity purification of proteins using IMAC is based on the interactions between a transition metal ion (Co2+, Ni2+, Cu2+, Zn2+) immobilized on a matrix and specific amino acid side chains such as histidine which exhibits the strongest interaction [66]. All the purified samples were dialyzed into 50 mM Tris, pH 8.0, 150 mM NaCl, 1 mM TCEP.…”