A polyhistidine-tag is an amino acid motif in proteins that consists of at least six histidine (His) residues, often at the N- or C-terminus of the protein. It is also known as hexa histidine-tag, 6xHis-tag, His6 tag and by the trademarked name His-tag (registered by EMD Biosciences). The tag was invented by Roche, although the use of histidines and its vectors are distributed by Qiagen. Various purification kits for histidine-tagged proteins are available from Qiagen, Sigma, Thermo Scientific, GE Healthcare, Macherey-Nagel, Clontech, Bio-Rad, and others.
The use of the tag for academic users is unrestricted; however, commercial users must pay royalties to Roche. The original patent expired on 11 Feb 2003, and thus should be now public property; current claims to royalties are based on a much narrower set of more recent patents. Suitable tag sequences are available free for commercial use; for example, MK(HQ)6 may be used for enhanced expression in E. coli and tag removal. The total number of histidine residues may vary in the tag. The his-tag may also be followed by a suitable amino acid sequence that facilitates a removal of the polyhistidine-tag using endopeptidases. This extra sequence is not necessary if exopeptidases are used to remove N-terminal His-tags (e.g., Qiagen TAGZyme). Furthermore, exopeptidase cleavage may solve the unspecific cleavage observed when using endoprotease-based tag removal. Polyhistidine-tags are often used for affinity purification of genetically modified proteins.
Polyhistidine-tags are often used for affinity purification of polyhistidine-tagged recombinant proteins expressed in Escherichia coli  and other prokaryotic expression systems. Bacterial cells are harvested via centrifugation and the resulting cell pellet lysed either by physical means or by means of detergents and enzymes such as lysozyme. At this stage raw lysate contains the recombinant protein among many other proteins originating from the bacterial host. This mixture is incubated with an affinity resin containing bound bivalent nickel or cobalt ions, which are available commercially in different varieties. Nickel and cobalt have similar properties and as they are adjacent period 4 transition metals ((v. iron triad)). These resins are generally sepharose/agarose functionalised with a chelator, such as iminodiacetic acid (Ni<super>2++</super>-IDA) and nitrilotriacetic acid (Ni<super>2++</super>-NTA) for nickel and carboxylmethylaspartate (Co<super>2++</super>-CMA) for cobalt, which the polyhistidine-tag binds with micromolar affinity. The resin is then washed with phosphate buffer to remove proteins that do not specifically interact with the cobalt or nickel ion. With Ni-based methods, washing efficiency can be improved by the addition of 20 mM imidazole (proteins are usually eluted with 150-300 mM imidazole). Generally nickel-based resins have higher binding capacity, while cobalt-based resins offer the highest purity. The purity and amount of protein can be assessed by SDS-PAGE and Western blotting.
Affinity purification using a polyhistidine-tag usually results in relatively pure protein when the recombinant protein is expressed in prokaryotic organisms. Depending on downstream applications, including the purification of protein complexes to study protein interactions, purification from higher organisms such as yeasts or other eukaryotes may require a tandem affinity purification using two tags to yield higher purity. Alternatively, single-step purification using immobilized cobalt ions rather than nickel ions generally yields a substantial increase in purity and requires lower imidazole concentrations for elution of the his-tagged protein.
Polyhistidine-tagging is the option of choice for purifying recombinant proteins in denaturing conditions because its mode of action is dependent only on the primary structure of proteins. Generally for this sort of a technique, histidine binding is titrated using pH instead of imidazole binding—at a high pH, histidine binds to nickel or cobalt, but at low pH (~6 for cobalt and ~4 for nickel), histidine becomes protonated and is competed off of the metal ion. Compare this to antibody purification and GST purification, a prerequisite to which is the proper (native) folding of proteins involved.
Polyhistidine-tag columns retain several well known proteins as impurities. One of them is FKBP-type peptidyl prolyl isomerase, which appears around 25kDa (SlyD). Impurities are generally eliminated using a secondary chromatographic technique, or by expressing the recombinant protein in a SlyD-deficient E. coli strain. Alternatively cobalt-based resins do not bind SlyD from E. coli and can be used for a single-step purification .
Separating One from Two Polyhistidine Tags
Proteins with different numbers of polyhistidine tags elute differently from nickel-affinity resin. For proteins with a single hexahistidine tag, 75 mM imidazole enables elution from Ni-NTA, whereas for proteins with two hexahistidine tags, 100 mM imidazole is required for elution. This step-wise elution may be used to isolate specific protein assemblies from a mixture, such as defined heteromultimers (e.g. an AB heterodimer from a mixture including AA and BB homodimers, if only subunit B has a polyhistidine tag). Such an approach was used in isolation of monovalent streptavidin.
Polyhistidine-tagging can be used to detect protein-protein interactions in the same way as a pull-down assay. However, this technique is generally considered to be less sensitive, and also restricted by some of the more finicky aspects of this technique. For example, reducing conditions cannot be used, EDTA and many types of detergents cannot be used. Recent advances in dual polarisation interferometry is amenable to EDTA and a wider use of reagents, and the use of such site-specific tags greatly simplifies the direct measurement of associated conformational change.
Hexahistadine CyDye tags have also been developed. These use Nickel covalent coordination to EDTA groups attached to fluorophores in order to create dyes that attach to the polyhistidine tag. This technique has been shown to be effective for following protein migration and trafficking. There has also been recent discoveries that show this technique may be effective in order to measure distance via Fluorescent Resonance Energy Transfer.
Adding Polyhistidine Tags
The most common polyhistidine tags are formed of six histidine (6xHis tag) residues - which are added at the N-terminus preceded by Methionine or C-terminus before a stop codon, in the coding sequence of the protein of interest. The choice of the end where His-tag is added will depend mainly on the characteristics of the protein and the methods chosen to remove the tag. Some ends are buried inside the protein core and others are important for the protein function or structure. In those cases the choice is limited to the other end. On the other hand, most available exopeptidases can only remove the His-tag from the N-terminus; removing the tag from the C-terminus will require the use of other techniques.
There are two ways to add polyhistidines. The most simple is to insert the DNA encoding the protein in a vector encoding a His-tag so that it will be automatically attached to one of its ends (See picture). Another technique is to perform a PCR with primers that have repetitive histidine codons (CAT or CAC) right next to the START or STOP codon in addition to several (16 or more) bases from one end of the DNA encoding the protein to be tagged (see primer example).
Example of primer designed to add a 6xHis-tag using PCR. Eighteen bases coding six histidines are inserted right after the START codon or right before the STOP codon. At least 16 bases specific to the gene of interest are needed next to the His-tag. With 6 His, the protein will have an added 1 kDa of molecular weight. Note: often, a linker (such as gly-gly-gly or gly-ser-gly) is placed between the protein of interest and the 6 His tag. This is to prevent the polyhistidine tag from affecting the activity of the protein being tagged.
The polyhistidine-tag can also be used to detect the protein via anti-polyhistidine-tag antibodies or alternatively by in-gel staining (SDS-PAGE) with fluorescent probes bearing metal ions. This can be useful in subcellular localization, ELISA, western blotting or other immuno-analytical methods.
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