Polyhistidine-tag
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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, and by the trademarked name His-tag® (registered by EMD Biosciences). The tag was invented by Roche and its vectors and NTA (nitrilotriaceticacid) protein purification kits are distributed by Qiagen. The use of the tag for academic users is unrestricted, however commercial users have to pay royalties to Roche. Suitable tag sequences are available for free commercial use, like e.g., MK(HQ)6 for enhanced expression in E. coli and tag removal. The total number of histidine residues may vary in the tag allowing for customized tags. The his-tag may also be followed by a suitable amino acid sequence that facilitates a removal of the polyhistidine-tag using endoproteases. This extra sequence is not necessary if exopeptidases are used to remove N-terminal histags (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.
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Applications
Protein purification
Polyhistidine-tags are often used for affinity purification of polyhistidine-tagged recombinant proteins that are expressed in Escherichia coli <ref>Trends Biochem Sci. 1995 20(7):285-6 Purification of His-Tag fusion proteins from Escherichia coli</ref> or other prokaryotic expression systems. The bacterial cells are harvested by centrifugation and the resulting cell pellet can be lysed with detergents or enzymes such as lysozyme. The raw lysate contains at this stage the recombinant protein among several other proteins derived from the bacteria and are incubated with affinity media such as NTA-agarose or Talon resin. Both affinity media have metal ions bound to it, either nickel or cobalt to 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. The washing efficiency can be improved by the addition of 20 mM imidazole and proteins are then usually eluted with 150-300 mM imidazole. The purity and amount of protein can be assessed by SDS-PAGE and western blotting. The affinity purification using a polyhistidine-tag works usually fine, when the recombinant protein was expressed in procaryotic host organism, whereas the purification from higher organisms such as yeast, insect cell or other eukaryotics may require a tandem affinity purification<ref>Nature. 2002 415(6868):141-7 Functional organization of the yeast proteome by systematic analysis of protein complexes</ref> using two tags in special cases or for special purposes like the purification of protein complexes to study protein interactions.
Because its mechanism is only dependent on the primary structure of proteins, polyhistidine-tagging is the option of choice for purifying recombinant proteins in denaturing conditions. 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 but at low pH (~4) histidine becomes protonated and is competed off of the metal ion. Compare this to antibody purification and gst purification which require some protein be properly folded.
Polyhistidine-tag columns will retain several well known proteins as impurities. One of them is FKBP-type peptidyl prolyl isomerase, which appears around 25kDa (SlyD). These should be separated in any protein prep by using a secondary chromatographic technique.
Binding assays
Polyhistidine-tagging can be used to detect protein-protein interactions in the same way as a gst pulldown 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.
Detection
The polyhistidine-tag can also be used in detection of the protein via anti-polyhistidine-tag antibodies in gel staining (SDS-PAGE) with fluorescently labelled NTA. This can be useful in subcellular localization, ELISA, western blotting or other immuno-analytical methods.
Immobilization
The polyhistidine-tag can be ideally used for the immobilization of proteins on a surface such as on a NTA coated microtiter plate or on a protein array.
References
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