His-tag

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A simple gravity flow column for Ni -affinity chromatography. The sample and subsequent buffers are manually poured into the column and collected at the bottom end after flowing through the resin bed (in light blue at the base of the column). Simple column for Ni2+-affinity chromatography.jpg
A simple gravity flow column for Ni -affinity chromatography. The sample and subsequent buffers are manually poured into the column and collected at the bottom end after flowing through the resin bed (in light blue at the base of the column).

A polyhistidine-tag, best known by the trademarked name His-tag, is an amino acid motif in proteins that typically consists of at least six histidine (His) residues, often at the N- or C-terminus of the protein. It is also known as a hexa histidine-tag, 6xHis-tag, or His6 tag. The tag was invented by Roche, [1] although the use of histidines and its vectors are distributed by Qiagen. Various purification kits for histidine-tagged proteins are commercially available from multiple companies. [2]

Contents

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 that facilitates 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.

Principle

Three views of the X-ray structure of Ni(NTA)(H2O)2 Ni(NTA)(aq)23views.png
Three views of the X-ray structure of Ni(NTA)(H2O)2

Proteins can coordinate metal ions on their surface and it is possible to separate proteins using chromatography by making use of the difference in their affinity to metal ions. This is termed as immobilized metal ion affinity chromatography (IMAC), as originally introduced in 1975 under the name metal chelate affinity chromatography. [3] Subsequent studies have revealed that among amino acids constituting proteins, histidine is strongly involved in the coordination complex with metal ions. [4] Therefore, if a number of histidines are added to the end of the protein, the affinity of the protein for the metal ion is increased and this can be exploited to selectively isolate the protein of interest. When a protein with a His-tag is brought into contact with a carrier on which a metal ion such as nickel is immobilized, 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. The poly-histidine tagged protein can then be recovered by eluting it off the resin. [5]

Practical choices

Tag length

Examples of methods for adding polyhistidine tags. (A) The polyhistidine tag is added by inserting the DNA encoding a protein of interest in a vector that has the tag ready to fuse at the C-terminus. (B) The polyhistidine tag is added using primers containing the tag coding sequence as an overhang on the forward primer. After PCR amplification, the tag is present on the N-terminus of the gene, which can then be sub-cloned into an expression vector. His-tag.png
Examples of methods for adding polyhistidine tags. (A) The polyhistidine tag is added by inserting the DNA encoding a protein of interest in a vector that has the tag ready to fuse at the C-terminus. (B) The polyhistidine tag is added using primers containing the tag coding sequence as an overhang on the forward primer. After PCR amplification, the tag is present on the N-terminus of the gene, which can then be sub-cloned into an expression vector.

Polyhistidine tags most commonly consist of six histidine residues. Tags with up to twelve histidine residues or dual tags attached via short linker are not uncommon though and may improve purification results by enhancing binding to the affinity resin, allowing for increased stringency of washing and separation from endogenous proteins. [6] [7] [8] The tag can be added to a gene of interest using methods common to most purification tags. The most basic method is to subclone the gene of interest into a vector containing a polyhistidine tag sequence. Many vectors for use with various expression systems are available with polyhistidine tags in a variety of positions and with differing protease cleavage sites, other tags etc. [9] However, if an appropriate vector is unavailable or the tag needs to be inserted at a location other than the proteins N- or C-terminus, the gene of interest can be either directly synthesised containing a polyhistidine tag sequence or various methods based on PCR can be used to add the tag to a gene. A common approach is to add the coding sequence for the polyhistidine tag to the PCR primers as an overhang. [10] [11]

Tag position

Most commonly, a polyhistidine tag is fused at the N-terminus or C-terminus of a protein and is attached via a short flexible linker, which may contain a protease cleavage site. [10] [9] Less commonly, tags can be added at both the N- and C-termini or inserted at an intermediate part of a protein, such as within an exposed loop. [12] [8] The choice of tag position depends on the properties of each protein and the chosen purification strategy; it may be necessary to test multiple constructs with the tag at different positions. [6] [10] Although polyhistidine tags are considered to typically not alter the properties of a protein, it has been demonstrated that addition of the tag can cause unwanted effects, such as influencing the protein's oligomeric state. [13]

Carrier matrices

Various carrier matrices bound to a solid resin support are on the market and these can be subsequently charged with a metal cation. Derivatives of iminodiacetic acid (IDA) and nitrilotriacetic acid (NTA) are most frequently used for this purpose, with differing matrices having certain advantages and disadvantages for various applications. [14]

Metal ions

Several metal cations have high affinities for imidazole, the functional group of the His-tag. Divalent cation M2+ (M = Mn, Fe, Co, Ni, Cu, Zi etc) transition metal imidazole complexes are most frequently used for this purpose. The choice of cation is generally a compromise between binding capacity and purity. Nickel is often used as it offers a good balance between these factors, while cobalt can be used when it is desired to increase the purity of purification as it has less affinity for endogenous proteins; binding capacity however is lower compared with nickel. [14] [10]

Elution method

In order to elute His-tagged protein from the carrier there are several potential methods, which can be used in combination if necessary. In order to avoid denaturation of proteins, it is generally desirable to use as mild a method as possible.

  • 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 typically used at a concentration of 150 - 500 mM for elution. Histidine or histamine can also 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 such as EDTA is added, the protein is detached from the carrier because the metal ion immobilized on the carrier is lost.

Applications

Protein purification

Polyhistidine-tags are often used for affinity purification of polyhistidine-tagged recombinant proteins expressed in Escherichia coli or other expression systems. Typically, 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 or any combination of these. At this stage, the lysate contains the recombinant protein among many endogenous proteins originating from the host cells. The lysate is exposed to affinity resin bound to a carrier matrix coupled with a divalent cation, either by direct addition of resin (batch binding) or by passing over a resin bed in a column format. The resin is then washed with buffer to remove proteins that do not specifically interact with bound cation and the protein of interest is eluted off the resin using buffer containing a high concentration of imidazole or a lowered pH. The purity and amount of protein can be assessed by methods such SDS-PAGE and Western blotting. [14] [10] [15]

Affinity purification using a polyhistidine-tag usually results in relatively pure protein. Protein purity can be improved by the addition of a low (20-40 mM) concentration of imidazole to the binding and/or wash buffers. However, depending on the requirements of the downstream application, further purification steps using methods such as ion exchange or size exclusion chromatography may be required. IMAC resins typically retain several prominent endogenous proteins as impurities. In E. coli for instance, a prominent example is FKBP-type peptidyl prolyl isomerase, which appears around 25 kDa on SDS-PAGE. These impurities can be eliminated using additional purification steps or by expressing the recombinant protein in a deficient strain of cells. Alternatively, cobalt charged IMAC resins which have less affinity for endogenous proteins can be used. [16] [10] [14] [17]

Binding assays

Polyhistidine-tagging can be used to detect protein-protein interactions in the same way as a pull-down assay. Polyhistidine tagging has several advantages over other tags commonly used for pull-down assays, including its small size, few naturally occurring proteins binding to the carrier matrices and the increased stability of the carrier matrix over monoclonal antibody matrices. [18]

Fluorescent tags

Hexahistadine CyDye tags have been developed, which 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 useful for following protein migration and trafficking and may be effective for measuring distance via Förster resonance energy transfer. [19]

Fluorohistidine tags

A polyfluorohistidine tag has been reported for use in in vitro translation systems. [20] In this system, an expanded genetic code 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.[ citation needed ]

Detection

The polyhistidine-tag can also be used for detecting a protein via anti-polyhistidine-tag antibodies, which can be useful for subcellular localization, ELISA, western blotting and other immuno-analytical methods. Alternatively, in-gel staining of SDS-PAGE or native-PAGE gels with fluorescent probes bearing metal ions can be used for detection of a polyhistidine tagged protein. [21]

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. [22]

HAT tag

The HAT tag is a peptide tag (KDHLIHNVHKEEHAHAHNK) derived from chicken lactate dehydrogenase, and is more likely to be a soluble protein with no bias in charge distribution compared to the His tag. [23] 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

Related Research Articles

In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent called the mobile phase, which carries it through a system on which a material called the stationary phase is fixed. Because the different constituents of the mixture tend to have different affinities for the stationary phase and are retained for different lengths of time depending on their interactions with its surface sites, the constituents travel at different apparent velocities in the mobile fluid, causing them to separate. The separation is based on the differential partitioning between the mobile and the stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation.

<span class="mw-page-title-main">Histidine</span> Chemical compound

Histidine (symbol His or H) is an essential amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated –NH3+ form under biological conditions), a carboxylic acid group (which is in the deprotonated –COO− form under biological conditions), and an imidazole side chain (which is partially protonated), classifying it as a positively charged amino acid at physiological pH. Initially thought essential only for infants, it has now been shown in longer-term studies to be essential for adults also. It is encoded by the codons CAU and CAC.

<span class="mw-page-title-main">Membrane protein</span> Proteins that are part of, or interact with, biological membranes

Membrane proteins are common proteins that are part of, or interact with, biological membranes. Membrane proteins fall into several broad categories depending on their location. Integral membrane proteins are a permanent part of a cell membrane and can either penetrate the membrane (transmembrane) or associate with one or the other side of a membrane. Peripheral membrane proteins are transiently associated with the cell membrane.

Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the specification of the function, structure and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Ideally, to study a protein of interest, it must be separated from other components of the cell so that contaminants will not interfere in the examination of the protein of interest's structure and function. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity and biological activity. The pure result may be termed protein isolate.

<span class="mw-page-title-main">Imidazole</span> Chemical compound

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-adjacent nitrogen atoms in meta-substitution.

Affinity chromatography is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another substance. The specific type of binding interaction depends on the biomolecule of interest; antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid binding interactions are frequently exploited for isolation of various biomolecules. Affinity chromatography is useful for its high selectivity and resolution of separation, compared to other chromatographic methods.

<span class="mw-page-title-main">Ion chromatography</span> Separates ions and polar molecules

Ion chromatography is a form of chromatography that separates ions and ionizable polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including small inorganic anions, large proteins, small nucleotides, and amino acids. However, ion chromatography must be done in conditions that are one pH unit away from the isoelectric point of a protein.

Protein tags are peptide sequences genetically grafted onto a recombinant protein. Tags are attached to proteins for various purposes. They can be added to either end of the target protein, so they are either C-terminus or N-terminus specific or are both C-terminus and N-terminus specific. Some tags are also inserted at sites within the protein of interest; they are known as internal tags.

FLAG-tag, or FLAG octapeptide, or FLAG epitope, is a peptide protein tag that can be added to a protein using recombinant DNA technology, having the sequence DYKDDDDK. It is one of the most specific tags and it is an artificial antigen to which specific, high affinity monoclonal antibodies have been developed and hence can be used for protein purification by affinity chromatography and also can be used for locating proteins within living cells. FLAG-tag has been used to separate recombinant, overexpressed protein from wild-type protein expressed by the host organism. FLAG-tag can also be used in the isolation of protein complexes with multiple subunits, because FLAG-tag's mild purification procedure tends not to disrupt such complexes. FLAG-tag-based purification has been used to obtain proteins of sufficient purity and quality to carry out 3D structure determination by x-ray crystallography.

<span class="mw-page-title-main">Nitrilotriacetic acid</span> Chemical compound

Nitrilotriacetic acid (NTA) is the aminopolycarboxylic acid with the formula N(CH2CO2H)3. It is a colourless solid. Its conjugate base nitrilotriacetate is used as a chelating agent for Ca2+, Co2+, Cu2+, and Fe3+.

Phosphoproteomics is a branch of proteomics that identifies, catalogs, and characterizes proteins containing a phosphate group as a posttranslational modification. Phosphorylation is a key reversible modification that regulates protein function, subcellular localization, complex formation, degradation of proteins and therefore cell signaling networks. With all of these modification results, it is estimated that between 30–65% of all proteins may be phosphorylated, some multiple times. Based on statistical estimates from many datasets, 230,000, 156,000 and 40,000 phosphorylation sites should exist in human, mouse, and yeast, respectively.

<span class="mw-page-title-main">Meir Wilchek</span> Israeli biochemist (born 1935)

Meir Wilchek is an Israeli biochemist. He is a professor at the Weizmann Institute of Science.

The Strep-tag system is a method which allows the purification and detection of proteins by affinity chromatography. The Strep-tag II is a synthetic peptide consisting of eight amino acids (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys). This peptide sequence exhibits intrinsic affinity towards Strep-Tactin, a specifically engineered streptavidin, and can be N- or C- terminally fused to recombinant proteins. By exploiting the highly specific interaction, Strep-tagged proteins can be isolated in one step from crude cell lysates. Because the Strep-tag elutes under gentle, physiological conditions, it is especially suited for generation of functional proteins.

The Streptavidin-Binding Peptide (SBP)-Tag is a 38-amino acid sequence that may be engineered into recombinant proteins. Recombinant proteins containing the SBP-Tag bind to streptavidin and this property may be utilized in specific purification, detection or immobilization strategies.

In the medical field of immunology, nanoCLAMP affinity reagents are recombinant 15 kD antibody mimetic proteins selected for tight, selective and gently reversible binding to target molecules. The nanoCLAMP scaffold is based on an IgG-like, thermostable carbohydrate binding module family 32 (CBM32) from a Clostridium perfringens hyaluronidase. The shape of nanoCLAMPs approximates a cylinder of approximately 4 nm in length and 2.5 nm in diameter, roughly the same size as a nanobody. nanoCLAMPs to specific targets are generated by varying the amino acid sequences and sometimes the length of three solvent exposed, adjacent loops that connect the beta strands making up the beta-sandwich fold, conferring binding affinity and specificity for the target.

<span class="mw-page-title-main">IBA Lifesciences</span>

IBA Lifesciences is a biotechnology company providing products and custom specific services for life science applications in academia and industry worldwide. IBA focusses on two business segments: cell selection and protein purification.

<span class="mw-page-title-main">Metal-binding protein</span>

Metal-binding proteins are proteins or protein domains that chelate a metal ion.

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Dye-ligand affinity chromatography is one of the Affinity chromatography techniques used for protein purification of a complex mixture. Like general chromatography, but using dyes to apply on a support matrix of a column as the stationary phase that will allow a range of proteins with similar active sites to bind to, refers to as pseudo-affinity. Synthetic dyes are used to mimic substrates or cofactors binding to the active sites of proteins which can be further enhanced to target more specific proteins. Follow with washing, the process of removing other non-target molecules, then eluting out target proteins out by changing pH or manipulate the salt concentration. The column can be reused many times due to the stability of immobilized dyes. It can carry out in a conventional way by using as a packed column, or in high-performance liquid chromatography (HPLC) column.

<span class="mw-page-title-main">Transition metal imidazole complex</span>

A transition metal imidazole complex is a coordination complex that has one or more imidazole ligands. Complexes of imidazole itself are of little practical importance. In contrast, imidazole derivatives, especially histidine, are pervasive ligands in biology where they bind metal cofactors.

References

  1. ↑ Hochuli E, Bannwarth W, Döbeli H, Gentz R, Stüber D (1988). "Genetic Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent". Bio/Technology. 6 (11): 1321–5. doi:10.1038/nbt1188-1321. S2CID   9518666. INIST   7229670.
  2. ↑ The use of the tag for academic users was unrestricted; however, commercial users had to pay royalties to Roche. The original patent expired on 11 Feb 2003, and is 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.
  3. ↑ Porath J, Carlsson J, Olsson I, Belfrage G (December 1975). "Metal chelate affinity chromatography, a new approach to protein fractionation". Nature. 258 (5536): 598–599. Bibcode:1975Natur.258..598P. doi:10.1038/258598a0. PMID   1678. S2CID   4271836.
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  5. ↑ "His-tag: The timeless standard for protein purification". Cube Biotech Knowledge Site. Archived from the original on 15 December 2021. Retrieved December 2, 2021.
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  9. 1 2 Singh MI, Jain V (2013-05-15). "Tagging the expressed protein with 6 histidines: rapid cloning of an amplicon with three options". PLOS ONE. 8 (5): e63922. Bibcode:2013PLoSO...863922S. doi: 10.1371/journal.pone.0063922 . PMC   3655076 . PMID   23691118.
  10. 1 2 3 4 5 6 Bornhorst JA, Falke JJ (2000-01-01). "Purification of proteins using polyhistidine affinity tags". Applications of Chimeric Genes and Hybrid Proteins Part A: Gene Expression and Protein Purification. Methods in Enzymology. Vol. 326. Academic Press. pp. 245–254. doi:10.1016/s0076-6879(00)26058-8. PMC   2909483 . PMID   11036646.
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  12. ↑ Paul DM, Beuron F, Sessions RB, Brancaccio A, Bigotti MG (February 2016). "Internal (His)₆-tagging delivers a fully functional hetero-oligomeric class II chaperonin in high yield". Scientific Reports. 6 (1): 20696. Bibcode:2016NatSR...620696P. doi:10.1038/srep20696. PMC   4746591 . PMID   26856373.
  13. ↑ Majorek KA, Kuhn ML, Chruszcz M, Anderson WF, Minor W (October 2014). "Double trouble-Buffer selection and His-tag presence may be responsible for nonreproducibility of biomedical experiments". Protein Science. 23 (10): 1359–1368. doi:10.1002/pro.2520. PMC   4286991 . PMID   25044180.
  14. 1 2 3 4 Riguero V, Clifford R, Dawley M, Dickson M, Gastfriend B, Thompson C, et al. (October 2020). "Immobilized metal affinity chromatography optimization for poly-histidine tagged proteins". Journal of Chromatography A. 1629: 461505. doi: 10.1016/j.chroma.2020.461505 . PMID   32861092. S2CID   221373404.
  15. ↑ Hengen P (July 1995). "Purification of His-Tag fusion proteins from Escherichia coli". Trends in Biochemical Sciences. 20 (7): 285–286. doi:10.1016/S0968-0004(00)89045-3. PMID   7667882.
  16. ↑ Chen X, Nomani A, Patel N, Hatefi A (June 2017). "Production of low-expressing recombinant cationic biopolymers with high purity". Protein Expression and Purification. 134: 11–17. doi:10.1016/j.pep.2017.03.012. PMC   5479735 . PMID   28315745.
  17. ↑ Andersen KR, Leksa NC, Schwartz TU (November 2013). "Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification". Proteins. 81 (11): 1857–1861. doi:10.1002/prot.24364. PMC   4086167 . PMID   23852738.
  18. ↑ Louche A, Salcedo SP, Bigot S (2017), Journet L, Cascales E (eds.), "Protein–Protein Interactions: Pull-Down Assays", Bacterial Protein Secretion Systems: Methods and Protocols, Methods in Molecular Biology, New York, NY: Springer, vol. 1615, pp. 247–255, doi:10.1007/978-1-4939-7033-9_20, ISBN   978-1-4939-7033-9, PMID   28667618 , retrieved 2023-06-08
  19. ↑ Zhao C, Hellman LM, Zhan X, Bowman WS, Whiteheart SW, Fried MG (April 2010). "Hexahistidine-tag-specific optical probes for analyses of proteins and their interactions". Analytical Biochemistry. 399 (2): 237–245. doi:10.1016/j.ab.2009.12.028. PMC   2832190 . PMID   20036207.
  20. ↑ Ring CM, Iqbal ES, Hacker DE, Hartman MC, Cropp TA (May 2017). "Genetic incorporation of 4-fluorohistidine into peptides enables selective affinity purification". Organic & Biomolecular Chemistry. 15 (21): 4536–4539. doi:10.1039/C7OB00844A. PMC   6010304 . PMID   28517015.
  21. ↑ Raducanu VS, Isaioglou I, Raducanu DV, Merzaban JS, Hamdan SM (August 2020). "Simplified detection of polyhistidine-tagged proteins in gels and membranes using a UV-excitable dye and a multiple chelator head pair". The Journal of Biological Chemistry. 295 (34): 12214–12223. doi: 10.1074/jbc.ra120.014132 . PMC   7443479 . PMID   32647010.
  22. ↑ US 7176298,Tchaga GS, Jokhadze GG,"Polynucleotides encoding metal ion affinity peptides and related products",issued 13 February 2007, assigned to Takara Bio USA Inc.
  23. ↑ Chaga G, Bochkariov DE, Jokhadze GG, Hopp J, Nelson P (December 1999). "Natural poly-histidine affinity tag for purification of recombinant proteins on cobalt(II)-carboxymethylaspartate crosslinked agarose". Journal of Chromatography A. 864 (2): 247–256. doi:10.1016/S0021-9673(99)01008-0. PMID   10669292.
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