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A polyhistidine-tag is an
amino acid Amino acids are organic compound In chemistry, organic compounds are generally any chemical compounds that contain carbon-hydrogen or carbon-carbon chemical bond, bonds. Due to carbon's ability to Catenation, catenate (form chains with ot ...
motif in
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metabo ...
s that typically consists of at least six
histidine Histidine (symbol His or H) is an essential amino acid that is used in the biosynthesis of proteins. It contains an Amine, α-amino group (which is in the protonated –NH3+ form under Physiological condition, biological conditions), a carboxylic ...
(''His'') residues, often at the N- or C-terminus of the protein. It is also known as hexa histidine-tag, 6xHis-tag, His6 tag, by the US trademarked name HIS TAG (US Trademark serial number 74242707), and most commonly as His-Tag. The tag was invented by Roche, although the use of histidines and its vectors are distributed by
Qiagen QIAGEN N.V., the global corporate headquarter of the QIAGEN group, is located in Venlo, The Netherlands. Furthermore, European, American, and Asia regional headquarters are located in respectively Hilden, Germany, Maryland United States, and Sh ...
. Various purification kits for histidine-tagged proteins are available from
Qiagen QIAGEN N.V., the global corporate headquarter of the QIAGEN group, is located in Venlo, The Netherlands. Furthermore, European, American, and Asia regional headquarters are located in respectively Hilden, Germany, Maryland United States, and Sh ...
,
Sigma Sigma (; uppercase Σ, lowercase Letter case is the distinction between the Letter (alphabet), letters that are in larger uppercase or capitals (or more formally ''majuscule'') and smaller lowercase (or more formally ''minuscule'') in the wr ...
,
Thermo Scientific Thermo Fisher Scientific Inc. is an American supplier of scientific instrumentation, reagents and consumables, and software services. Based in Waltham, Massachusetts, Thermo Fisher was formed through the Mergers and acquisitions, merger of Ther ...
,
GE Healthcare GE HealthCare is a subsidiary of American Multinational corporation, multinational conglomerate General Electric incorporated in New York (state), New York and headquartered in Chicago, Illinois. As of 2017, it is a manufacturer and distributor o ...
, Macherey-Nagel
Cube Biotech
Clontech,
Bio-Rad Bio-Rad Laboratories, Inc. is an American developer and manufacturer of specialized technological products for the life science Life is a quality that distinguishes matter that has biological processes, such as Cell signaling, signal ...


and others. 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 from as low as two, to as high as 10 or more His residues. N- or C-terminal His-tags may also be followed or preceded, respectively, by a suitable
amino acid sequence Protein primary structure is the Biomolecular structure#Primary structure, linear sequence of amino acids in a peptide or protein. By convention, the Protein structure#Primary structure, primary structure of a protein is reported starting from the ...
that facilitates removal of the polyhistidine-tag using
endopeptidases Endopeptidase or endoproteinase are Proteolysis, proteolytic peptidases that break peptide bonds of nonterminal amino acids (i.e. within the molecule), in contrast to exopeptidases, which break peptide bonds from end-pieces of terminal amino acids. ...
. 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 Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of Genetic engineering techniques, technologies used to change the gene ...
proteins.


Principle

In general, proteins possess more or less the ability to coordinate metal ions on their surface, and it is possible to separate proteins by chromatography making use of the difference in their affinity. This is the immobilized metal ion affinity chromatography announced in 1975. Subsequent studies have revealed that among amino acids constituting proteins, histidine is strongly involved in the coordinate bond with metal ions. Therefore, if a number of histidines are added to the end of the protein by genetic engineering, the affinity of the protein for the metal ion is remarkably increased and the basic idea is that purification can be easily carried out. When a protein having a His-tag is brought into contact with a carrier on which a metal ion such as nickel is immobilized under the condition of pH 8 or higher, the histidine residue chelates the metal ion and binds to the carrier. Since other proteins do not bind to the carrier or bind only very weakly, they can be removed by washing the carrier with an appropriate buffer. Thereafter, by removing imidazole or the like from the carrier, it is possible to recover the protein having the His-tag with high purity.


Practical choice


Carrier

Various carriers such a
Ni - NTA agarose
(nickel - nitrilotriacetic acid) are on the market. It is packed in a column and used in combination with centrifugation and magnetic separation in a test tube.


Metal ions

Several metal cations have high affinities for imidazole, the function group of the His-tag. Transition metal imidazole complexes are well known for M2+ (M = Mn, Fe, Co, Ni, Cu). Nickel is often used for ordinary purposes, and cobalt is used when it is desired to increase the purity of purification.


Elution method

In order to elute His-tagged protein from the carrier, there is a plurality of methods as follows and it will be used properly according to the purpose. In order to avoid denaturation of proteins, it is desirable to have as mild as possible, and imidazole addition is often used from this viewpoint.


Competition with analogs

For releasing the His-tagged protein from the carrier, a compound is used that has a structure similar to the His-tag and which also forms a coordination complex with the immobilized metal ions. Such a compound added to the His-tagged protein on the carrier competes with the protein for the immobilized metal ions. The compound added at high concentration replaces virtually all carrier-bound protein which is thus eluted from the carrier. Imidazole is the side chain of histidine and is frequently used for this elution method at a concentration of 150 mM or more. Also histidine and histamine can be used.


Decrease in pH

When the pH decreases, the histidine residue is protonated and can no longer coordinate the metal tag, allowing the protein to be eluted. When nickel is used as the metal ion, it is eluted at around pH 4 and cobalt at around pH 6.


Removal of metal ions

When a strong chelating agent is added, the protein is detached from the carrier because the metal ion immobilized on the carrier is lost. EDTA is used exclusively.


Applications


Protein purification

Polyhistidine-tags are often used for affinity purification of polyhistidine-tagged recombinant proteins expressed in ''
Escherichia coli ''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative bacteria, Gram-negative, Facultative anaerobic organism, facultative anaer ...
'' and other prokaryotic expression systems. Bacterial cells are harvested via
centrifugation Centrifugation is a mechanical process which involves the use of the centrifugal force to separate particles from a solution according to their size, shape, density, medium viscosity and rotor speed. The denser components of the mixture migrate ...
and the resulting cell pellet lysed either by physical means or by means of detergents and
enzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different molecule ...
s such as
lysozyme Lysozyme (EC 3.2.1.17, muramidase, ''N''-acetylmuramide glycanhydrolase; systematic name peptidoglycan ''N''-acetylmuramoylhydrolase) is an antimicrobial enzyme produced by animals that forms part of the innate immune system. It is a glycoside ...
or any combination of these. At this stage, raw lysate contains the recombinant protein among many other proteins originating from the bacterial host. This mixture is incubated with commercially available affinity resin containing bound
divalent In chemistry, the valence (US spelling) or valency (British spelling) of an chemical element, element is the measure of its combining capacity with other atoms when it forms chemical compounds or molecules. Description The combining capacity, ...
nickel Nickel is a chemical element with Chemical symbol, symbol Ni and atomic number 28. It is a silvery-white lustrous metal with a slight golden tinge. Nickel is a hard and Ductility, ductile transition metal. Pure nickel is chemically reactive bu ...
or
cobalt Cobalt is a chemical element with the Symbol (chemistry), symbol Co and atomic number 27. As with nickel, cobalt is found in the Earth's crust only in a chemically combined form, save for small deposits found in alloys of natural meteoric iron. T ...
ions (both metals have similar properties as they are neighbouring transition metals). These resins are generally sepharose/agarose functionalized with a chelator, such as
iminodiacetic acid Iminodiacetic acid is the organic compound with the formula HN(CH2CO2H)2, often abbreviated to IDA. A white solid, the compound is a dicarboxylic acid amine (the nitrogen atom forms a secondary amino group, not an imino group as the name suggests) ...
(Ni-IDA) and
nitrilotriacetic acid Nitrilotriacetic acid (NTA) is the aminopolycarboxylic acid with the formula N(CH2CO2H)3. It is a colourless solid that is used as a chelating agent Chelation is a type of bonding of ions and molecules to metal ions. It involves the formatio ...
(Ni-NTA) for nickel and carboxyl-methyl aspartate (Co-CMA) for cobalt, all of which are bound by the polyhistidine-tag with micromolar affinity. For example Ernst Hochuli et al. reported in 1987 the coupling of the NTA ligand and Nickel-ions to
agarose Agarose is a heteropolysaccharide, generally extracted from certain Red algae, red seaweed. It is a linear polymer made up of the repeating unit of agarobiose, which is a disaccharide made up of D-galactose, D-galactose and 3,6-anhydro-L-galacto ...
beads. 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 Imidazole (ImH) is an organic compound with the formula C3N2H4. It is a white or colourless solid that is soluble in water, producing a mildly alkaline solution. In chemistry, it is an aromatic heterocycle, classified as a diazole, and has non-ad ...
(proteins are usually eluted with 150-300 mM imidazole, see section on the elution method above). Generally, nickel-based resins have a higher binding capacity, while cobalt-based resins offer higher purity. The purity and amount of protein can be assessed by
SDS-PAGE SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a Discontinuous electrophoresis, discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular m ...
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
yeast Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom (biology), kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are est ...
s or other
eukaryote Eukaryotes () are organisms whose Cell (biology), cells have a cell nucleus, nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the ...
s 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. For example, even when a recombinant protein forcibly expressed in ''E. coli'' produces an inclusion body and can not be obtained as a soluble protein, it can be purified with denaturation with urea or guanidine hydrochloride. 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. On the other hand, it is said that the His tag tends to aggregate and insolubilize more than other affinity tags. 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, comparing with nickel-based, cobalt-based resins have less affinity with SlyD from ''E. coli'', but in several cases, it is moderately helpful.


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 Streptavidin is a 66.0 (tetramer) Atomic mass unit, kDa protein purified from the bacterium ''Streptomyces avidinii''. Streptavidin Homotetramer, homo-tetramers have an extraordinarily high affinity for biotin (also known as vitamin B7 or vitam ...
.


Binding assays

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 Ethylenediaminetetraacetic acid (EDTA) is an aminopolycarboxylic acid with the formula H2N(CH2CO2H)2. This white, water-soluble solid is widely used to bind to iron (Fe2+/Fe3+) and calcium ions (Ca2+), forming water-soluble coordination com ...
and many types of detergents cannot be used. Recent advances in
dual polarisation interferometry Dual-polarization interferometry (DPI) is an analytical technique that probes molecular layers adsorbed to the surface of a Waveguide (optics), waveguide using the evanescent wave of a laser beam. It is used to measure the conformational change ...
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 In biochemistry, a conformational change is a change in the shape of a macromolecule, often induced by environmental factors. A macromolecule is usually flexible and dynamic. Its shape can change in response to changes in its environment or othe ...
.


Fluorescent tags

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.


Fluorohistidine tags

A polyfluorohistidine tag has been reported for use in ''in vitro'' translation systems. In this system, an
expanded genetic code An expanded genetic code is an artificially modified genetic code in which one or more specific codons have been re-allocated to encode an amino acid that is not among the 22 common naturally-encoded proteinogenic amino acids. The key prerequisit ...
is used in which histidine is replaced by 4-fluorohistidine. The fluorinated analog is incorporated into peptides via the relaxed substrate specificity of histidine-tRNA ligase and lowers the overall pKa of the tag. This allows for the selective enrichment of polyfluorohistidine tagged peptides in the presence of complex mixtures of traditional polyhistidine tags by altering the pH of the wash buffers.


Adding polyhistidine tags

The most common polyhistidine tags are formed of six histidine (6xHis tag) residues - which are added at the
N-terminus The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. Within a peptide, the amin ...
preceded by
Methionine Methionine (symbol Met or M) () is an essential amino acid in humans. As the precursor of other amino acids such as cysteine and taurine, versatile compounds such as SAM-e, and the important antioxidant glutathione, methionine plays a critical rol ...
or
C-terminus The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH). When the protein is ...
before a stop codon, in the coding sequence of the
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metabo ...
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. It is important to take into account that the computer simulation (by molecular dynamics) will help you to choose between options, for example, whether the His-tag must be digested or engineered to the N- or C-terminal. 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 /upload.wikimedia.org/wikipedia/commons/0/0c/His-tag.png (See picture) Another technique is to perform a PCR with primers that have repetitive histidine
codons The genetic code is the set of rules used by living cells to translate information encoded within genetic material ( DNA or RNA sequences of nucleotide triplets, or codons) into protein Proteins are large biomolecules and macromolecules ...
(CAT or CAC) right next to the
START Start can refer to multiple topics: *Takeoff, the phase of flight where an aircraft transitions from moving along the ground to flying through the air *Starting lineup in sports *Standing start, and rolling start, in an auto race Acronyms *Str ...
or
STOP codon In molecular biology (specifically protein biosynthesis), a stop codon (or termination codon) is a Genetic code, codon (nucleotide triplet within messenger RNA) that signals the termination of the translation (biology), translation process of the ...
in addition to several (16 or more) bases from one end of the DNA encoding the protein to be tagged (see primer example below). ''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. Often, a linker (such as gly-gly-gly or gly-ser-gly) is placed between the protein of interest and the 6 His tag in order to prevent the polyhistidine tag from affecting the activity of the protein being tagged.


Detection

The polyhistidine-tag can also be used to detect the protein via anti-polyhistidine-tag
antibodies An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform ...
or alternatively by in-gel staining (
SDS-PAGE SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a Discontinuous electrophoresis, discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular m ...
) with fluorescent probes bearing metal ions. This can be useful in
subcellular localization The cell (biology), cells of eukaryote, eukaryotic organisms are elaborately subdivided into functionally-distinct membrane-bound compartments. Some major constituents of eukaryotic cells are: extracellular space, Cell membrane, plasma membrane, cyt ...
,
ELISA The enzyme-linked immunosorbent assay (ELISA) (, ) is a commonly used analytical biochemistry assay, first described by Eva Engvall and Peter Perlmann in 1971. The assay uses a solid-phase type of enzyme immunoassay (EIA) to detect the presence ...
, western blotting or other immuno-analytical methods.


Immobilization

The polyhistidine-tag can be successfully used for the immobilization of proteins on a surface such as on a nickel- or cobalt-coated microtiter plate or on a protein array.


Similar tags


HQ tag

The HQ tag has alternating histidine and glutamine (HQHQHQ).


HN tag

The HN tag has alternating histidine and asparagine (HNHNHNHNHNHN) and is more likely to be presented on the protein surface than Histidine-only tags. The HN tag binds to the immobilized metal ion more efficiently than the His tag.


HAT tag

The HAT tag is a peptide tag (KDHLIHNVHKEEHAHAHNK) derived from chicken
lactate dehydrogenase Lactate dehydrogenase (LDH or LD) is an enzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enz ...
, and is more likely to be a soluble protein with no bias in charge distribution compared to the His tag. The arrangement of histidines in the HAT tag allows high accessibility compared to the His tag, and it binds efficiently to the immobilized metal ion.


See also

* Protein tag


References


External links


Ni - NTA affinity column
Protein Science Society Archive # 019/ Article in Japanese) {{DEFAULTSORT:Polyhistidine-Tag Biochemical separation processes Biochemistry detection methods Molecular biology techniques Protein structure