A polyhistidine-tag is an amino acid

Amino acid

Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...

 motif in protein

Protein

Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

s that consists of at least five histidine

Histidine

Histidine Histidine, an essential amino acid, has a positively charged imidazole functional group. It is one of the 22 proteinogenic amino acids. Its codons are CAU and CAC. Histidine was first isolated by German physician Albrecht Kossel in 1896. Histidine is an essential amino acid in humans...

 (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

Merck KGaA

Merck KGaA is a German chemical and pharmaceutical company. Merck, also known as “German Merck” and “Merck Darmstadt”, was founded in Darmstadt, Germany, in 1668, making it the world's oldest operating chemical and pharmaceutical company. The company was privately owned until going public in 1995...

). The tag was invented by Roche, although the use of histidines and its vectors are distributed by Qiagen

Qiagen

Qiagen is a provider of sample and assay technologies for molecular diagnostics, applied testing, academic and pharmaceutical research. Consolidated under the Dutch holding Qiagen N.V., the company operates more than 30 subsidiaries in over 18 countries. Qiagen’s shares are listed at the...

. Various purification kits for histidine-tagged proteins are available from Qiagen

Qiagen

Qiagen is a provider of sample and assay technologies for molecular diagnostics, applied testing, academic and pharmaceutical research. Consolidated under the Dutch holding Qiagen N.V., the company operates more than 30 subsidiaries in over 18 countries. Qiagen’s shares are listed at the...

, Sigma, Thermo Scientific, GE Healthcare

GE Healthcare

GE Healthcare is a division of GE Technology Infrastructure, which is itself a division of General Electric . It employs more than 46,000 people worldwide and is headquartered in Little Chalfont, Buckinghamshire, United Kingdom. GE Healthcare is the first GE business segment to be headquartered...

, Macherey-Nagel, Clontech, Bio-Rad

Bio-Rad

Bio-Rad Laboratories, Inc. , was founded in 1952 in Berkeley, California. The company was initially engaged in the development and production of specialty chemicals used in biochemical, pharmaceutical, and other life science research applications...

, and others.

The use of the tag for academic users is unrestricted; however, commercial users must pay royalties

Royalties

Royalties are usage-based payments made by one party to another for the right to ongoing use of an asset, sometimes an intellectual property...

 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

Affinity chromatography

Affinity chromatography is a method of separating biochemical mixtures and based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.-Uses:Affinity chromatography can be used to:...

 of genetically modified

Genetic engineering

Genetic engineering, also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest...

 proteins.

Protein purification

Polyhistidine-tags are often used for affinity purification of polyhistidine-tagged recombinant proteins expressed in Escherichia coli

Escherichia coli

Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...

and other prokaryotic expression systems. Bacterial cells are harvested via centrifugation

Centrifugation

Centrifugation is a process that involves the use of the centrifugal force for the sedimentation of mixtures with a centrifuge, used in industry and in laboratory settings. More-dense components of the mixture migrate away from the axis of the centrifuge, while less-dense components of the mixture...

 and the resulting cell pellet lysed either by physical means or by means of detergents and enzyme

Enzyme

Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...

s such as lysozyme

Lysozyme

Lysozyme, also known as muramidase or N-acetylmuramide glycanhydrolase, are glycoside hydrolases, enzymes that damage bacterial cell walls by catalyzing hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between...

. At this stage raw lysate contains the recombinant protein among many other proteins originating from the bacterial host. This mixture is incubated with affinity media such as Ni Sepharose, NTA-agarose, His60 Ni, HisPur resin, or TALON resin. Affinity media contain bound metal ions, either nickel

Nickel

Nickel is a chemical element with the chemical symbol Ni and atomic number 28. It is a silvery-white lustrous metal with a slight golden tinge. Nickel belongs to the transition metals and is hard and ductile...

 or cobalt

Cobalt

Cobalt is a chemical element with symbol Co and atomic number 27. It is found naturally only in chemically combined form. The free element, produced by reductive smelting, is a hard, lustrous, silver-gray metal....

 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. Washing efficiency can be improved by the addition of 20 mM imidazole

Imidazole

Imidazole is an organic compound with the formula C3H4N2. This aromatic heterocyclic is a diazole and is classified as an alkaloid. Imidazole refers to the parent compound, whereas imidazoles are a class of heterocycles with similar ring structure, but varying substituents...

 (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

SDS-PAGE

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, describes a collection of related techniques widely used in biochemistry, forensics, genetics and molecular biology to separate proteins according to their electrophoretic mobility...

 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

Yeast

Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi, with 1,500 species currently described estimated to be only 1% of all fungal species. Most reproduce asexually by mitosis, and many do so by an asymmetric division process called budding...

s or other eukaryote

Eukaryote

A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...

s may require a tandem affinity purification

Tandem Affinity Purification

Tandem affinity purification is a technique for studying protein-protein interactions. It involves creating a fusion protein with a designed piece, the TAP tag, on the end...

 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

Glutathione S-transferase

Enzymes of the glutathione S-transferase family are composed of many cytosolic, mitochondrial, and microsomal proteins. GSTs are present in eukaryotes and in prokaryotes, where they catalyze a variety of reactions and accept endogenous and xenobiotic substrates.GSTs can constitute up to 10% of...

 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 http://www.biosciencetechnology.com/Archive/01/Cobalt-based-Protein-Purification-Resin-Does-Not-Bind-E--coli-SlyD,-A-Common-Contaminant-in-Ni-NTA-IMAC/.

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

EDTA

Ethylenediaminetetraacetic acid, widely abbreviated as EDTA , is a polyamino carboxylic acid and a colourless, water-soluble solid. Its conjugate base is named ethylenediaminetetraacetate. It is widely used to dissolve limescale. Its usefulness arises because of its role as a hexadentate ligand...

 and many types of detergents cannot be used. Recent advances in dual polarisation interferometry

Dual Polarisation Interferometry

Dual polarization interferometry is an analytical technique that can probe molecular scale layers adsorbed to the surface of a waveguide by using the evanescent wave of a laser beam confined to the waveguide...

 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

Conformational change

A macromolecule is usually flexible and dynamic. It can change its shape in response to changes in its environment or other factors; each possible shape is called a conformation, and a transition between them is called a conformational change...

.

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.

Adding Polyhistidine Tags

The most common polyhistidine tags are formed of six histidine (6xHis tag) residues - preceded by Methionine

Methionine

Methionine is an α-amino acid with the chemical formula HO2CCHCH2CH2SCH3. This essential amino acid is classified as nonpolar. This amino-acid is coded by the codon AUG, also known as the initiation codon, since it indicates mRNA's coding region where translation into protein...

 - which are added at the C-terminal  or N-terminal of the protein

Protein

Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

 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-terminal, therefore removing the tag from the C-terminal 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

Primer (molecular biology)

A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. They are required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA...

 that have repetitive histidine codons (CAT or CAC) right next to the START

Start codon

The start codon is generally defined as the point, sequence, at which a ribosome begins to translate a sequence of RNA into amino acids.When an RNA transcript is "read" from the 5' carbon to the 3' carbon by the ribosome the start codon is the first codon on which the tRNA bound to Met,...

 or STOP codon

Stop codon

In the genetic code, a stop codon is a nucleotide triplet within messenger RNA that signals a termination of translation. Proteins are based on polypeptides, which are unique sequences of amino acids. Most codons in messenger RNA correspond to the addition of an amino acid to a growing polypeptide...

 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: oftentimes, 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.

Detection

The polyhistidine-tag can also be used to detect the protein via anti-polyhistidine-tag antibodies

Antibody

An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...

 or alternatively by in-gel staining (SDS-PAGE

SDS-PAGE

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, describes a collection of related techniques widely used in biochemistry, forensics, genetics and molecular biology to separate proteins according to their electrophoretic mobility...

) with fluorescent probes bearing metal ions. This can be useful in subcellular localization

Cell (biology)

The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....

, ELISA

ELISA

Enzyme-linked immunosorbent assay , is a popular format of a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay to detect the presence of a substance in a liquid sample."Wet lab" analytic biochemistry assays involves detection of an...

, 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 nickel or cobalt coated microtiter plate

Microtiter plate

A Microtiter plate or microplate or microwell plate, is a flat plate with multiple "wells" used as small test tubes. The microplate has become a standard tool in analytical research and clinical diagnostic testing laboratories...

 or on a protein array.