Biology:Polyhistidine-tag

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A simple gravity flow column for Ni2+-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 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 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,[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]

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 [citation needed]

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

Carrier matricies

Various carrier matricies 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 matricies having certain advantages and disadvantages for various applications.[6]

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.[6][7]

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 matix 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.[6][7][8]

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.[9][7][6][10]

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 occuring proteins binding to the carrier matricies and the increased stability of the carrier matrix over monoclonal antibody matricies.[11]

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.[12]

Fluorohistidine tags

A polyfluorohistidine tag has been reported for use in in vitro translation systems.[13] 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.

Adding polyhistidine tags

Adding polyhistidine tags. (A) The His-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 His-tag is added using primers containing the tag, after a PCR reaction the tag gets fused to the N-terminus of the gene.

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. 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.[14]

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 below).[citation needed]

His-tag-primers.png

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.[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.[15]

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.[16]

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.[17] 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

References

  1. ↑ "Genetic Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent". Bio/Technology 6 (11): 1321–5. 1988. doi:10.1038/nbt1188-1321. 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. ↑ "Metal chelate affinity chromatography, a new approach to protein fractionation". Nature 258 (5536): 598–599. December 1975. doi:10.1038/258598a0. PMID 1678. Bibcode: 1975Natur.258..598P. 
  4. ↑ "Immobilized metal ion affinity chromatography". Protein Expression and Purification 3 (4): 263–281. August 1992. doi:10.1016/1046-5928(92)90001-D. PMID 1422221. 
  5. ↑ "His-tag: The timeless standard for protein purification". Cube Biotech Knowledge Site. https://cube-biotech.com/his-tag-the-timeless-standard-for-protein-purification#howwork. 
  6. ↑ 6.0 6.1 6.2 6.3 "Immobilized metal affinity chromatography optimization for poly-histidine tagged proteins". Journal of Chromatography. A 1629: 461505. October 2020. doi:10.1016/j.chroma.2020.461505. PMID 32861092. 
  7. ↑ 7.0 7.1 7.2 "Purification of proteins using polyhistidine affinity tags". Methods in Enzymology. Applications of Chimeric Genes and Hybrid Proteins Part A: Gene Expression and Protein Purification (Academic Press) 326: 245–254. 2000-01-01. doi:10.1016/s0076-6879(00)26058-8. PMID 11036646. 
  8. ↑ "Purification of His-Tag fusion proteins from Escherichia coli". Trends in Biochemical Sciences 20 (7): 285–286. July 1995. doi:10.1016/S0968-0004(00)89045-3. PMID 7667882. 
  9. ↑ "Production of low-expressing recombinant cationic biopolymers with high purity". Protein Expression and Purification 134: 11–17. June 2017. doi:10.1016/j.pep.2017.03.012. PMID 28315745. 
  10. ↑ "Optimized E. coli expression strain LOBSTR eliminates common contaminants from His-tag purification". Proteins 81 (11): 1857–1861. November 2013. doi:10.1002/prot.24364. PMID 23852738. 
  11. ↑ "Protein–Protein Interactions: Pull-Down Assays" (in en), Bacterial Protein Secretion Systems: Methods and Protocols, Methods in Molecular Biology (New York, NY: Springer): pp. 247–255, 2017, doi:10.1007/978-1-4939-7033-9_20, ISBN 978-1-4939-7033-9 
  12. ↑ "Hexahistidine-tag-specific optical probes for analyses of proteins and their interactions". Analytical Biochemistry 399 (2): 237–245. April 2010. doi:10.1016/j.ab.2009.12.028. PMID 20036207. 
  13. ↑ "Genetic incorporation of 4-fluorohistidine into peptides enables selective affinity purification". Organic & Biomolecular Chemistry 15 (21): 4536–4539. May 2017. doi:10.1039/C7OB00844A. PMID 28517015. 
  14. ↑ "Lactobacillus plantarum WCFS1 β-Fructosidase: Evidence for an Open Funnel-Like Channel Through the Catalytic Domain with Importance for the Substrate Selectivity". Applied Biochemistry and Biotechnology 180 (6): 1056–1075. November 2016. doi:10.1007/s12010-016-2152-2. PMID 27295039. 
  15. ↑ "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. August 2020. doi:10.1074/jbc.ra120.014132. PMID 32647010. 
  16. ↑ U.S. Patent 7,176,298
  17. ↑ "Natural poly-histidine affinity tag for purification of recombinant proteins on cobalt(II)-carboxymethylaspartate crosslinked agarose". Journal of Chromatography. A 864 (2): 247–256. December 1999. doi:10.1016/S0021-9673(99)01008-0. PMID 10669292. 

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