Polyhistidine affinity tags are small enough to be incorporated easily into any expression vector. 12 If necessary, the affinity tag can be removed by use of a protease cleavage site inserted between the tag and the protein. Thus, the affinity tag usually does not need to be removed following protein purification. In principle, it is possible that the affinity tag may interfere with protein activity, although the relatively small size and charge of the polyhistidine affinity tag ensure that protein activity is rarely affected. Moving the affinity tag to the opposite terminus of the protein or carrying out the purification under denaturing conditions often resolves this problem. A potential problem is inaccessibility of the protein tag to the immobilized metal due to occlusion of the tag in the folded protein. Optimal placement of the tag is protein specific. Polyhistidine affinity tags are commonly placed on either the N or the C terminus of recombinant proteins.
#AFFINITY CHROMATOGRAPHY TRIAL#
In general, a six histidine tag is an appropriate choice for the first trial when adding a novel polyhistidine tag to a protein. 11 Still, it is advisable to use the smallest number of histidine residues as required for efficient purification to minimize possible perturbation of protein function. In some cases the use of longer polyhistidine tags has resulted in increased purity due to the ability to use more stringent washing steps. Whereas tags of six histidine residues are generally long enough to yield high-affinity interactions with the matrix, both shorter and longer affinity tags have been used successfully. Incorporation of the Polyhistidine Affinity TagĪffinity tags consisting of six polyhistidine residues are commonly used in IMAC. In such cases, either the use of a different affinity tag or the use of the polyhistidine tag in conjunction with additional purification techniques should be considered. 10 However, this purification method may not be sufficient for tagged proteins expressed at low levels that require significantly greater than 100-fold enrichment or for the preparation of highly homogeneous protein samples. 5, 6 Purification using polyhistidine tags has been carried out successfully using a number of expression systems, including Escherichia coli, 7 Saccharomyces cerevisiae, 8 mammalian cells, 9 and baculovirus-infected insect cells. 4 Affinity-tagged protein purities can be achieved at up to 95% purity by IMAC in high yield. IMAC is a versatile method that can be utilized to rapidly purify polyhistidine affinity-tagged proteins, resulting in 100-fold enrichments in a single purification step.
#AFFINITY CHROMATOGRAPHY FREE#
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. Peptides containing sequences of consecutive histidine residues are efficiently retained on IMAC column matrices. 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. 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. 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. 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. The expression and subsequent purification of recombinant proteins are widely employed in biochemical studies.
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