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chemistry

His-tag

His-tag is a chemistry topic covered in the lgStudy science library. This page brings together a partial reference excerpt, illustrations, worked examples, real-world applications and a short study plan, so you can understand His-tag rather than just read about it. In short: 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.

His-tag — main illustration
His-tag — illustration

Key takeaways

  • His-tag belongs to chemistry; place it in that map before memorising details.
  • Learn the definition first, then one example that makes the definition concrete.
  • Connect His-tag to a quantity you can measure, compute or draw — that is where exam questions come from.
  • Reproduce the core statement of His-tag from memory before moving on to harder problems.

Reference excerpt

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, 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. 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

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. Subsequent studies have revealed that among amino acids constituting proteins, histidine is strongly involved in the coordination complex with metal ions. 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.

Practical choices

Tag length

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. 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. 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.

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. 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. 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. 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.

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.

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, Zn 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.

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 100 - 500 mM for elution. Histidine 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

… excerpt ends here. Continue reading the full article.

Illustrations

His-tag: 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 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).
His-tag: Three views of the X-ray structure of Ni(NTA)(H2O)2 [citation needed]
Three views of the X-ray structure of Ni(NTA)(H2O)2 [citation needed]
His-tag: 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.
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.

Worked examples

Example 1 — a first encounter with His-tag

Start with the simplest possible case. Write down what His-tag claims or describes in one sentence, then invent the smallest concrete situation in which that sentence is true. In chemistry, the smallest case is usually a single object, a single equation or a single measurement. Check that every symbol or term in your sentence has a meaning in that case.

Example 2 — changing one variable

Take the situation from Example 1 and change exactly one quantity: double it, halve it, or set it to zero. Predict what should happen to His-tag before you calculate. Comparing your prediction with the result is the fastest way to find out whether you understand the idea or only the words.

Example 3 — an exam-style question

Typical questions about His-tag ask you to (a) state it precisely, (b) apply it to given data, and (c) explain a limitation. Practise writing all three answers in under five minutes; the third part is what separates a full-mark answer from an average one.

Applications of His-tag

In research
His-tag appears in chemistry research whenever the underlying quantities have to be modelled precisely. Papers usually cite it as a starting assumption and then explore where it breaks down.
In technology and industry
Engineering practice reuses His-tag in design rules, simulations and safety margins. Knowing the idea lets you read a specification sheet and understand why the numbers look the way they do.
In the classroom
His-tag is common in secondary-school and first-year university syllabi. It links to neighbouring topics Biochemical separation processes, Biochemistry detection methods, Molecular biology techniques, so understanding it makes those chapters shorter.
In everyday life
Look for His-tag outside the textbook — in sport, cooking, traffic, electronics or the sky above you. An example you found yourself is remembered far longer than one you were given.

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How to study His-tag in 20 minutes

  1. Read the reference excerpt below once, without taking notes.
  2. Close the page and write down what His-tag means in your own words.
  3. Compare your version with the excerpt and mark what you missed.
  4. Work through the three examples above with pen and paper.
  5. Explain His-tag out loud to somebody else — or to Teacher Smith in the lgStudy chat.

Frequently asked questions

What is His-tag in simple terms?

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.

Why does His-tag matter?

Because it connects several chemistry ideas at once: it gives you a definition you can apply, a quantity you can calculate, and a way to check whether a result is plausible.

How should I study His-tag?

Read the excerpt, restate it from memory, then work through the examples and applications listed on this page. The five-step study plan above takes about twenty minutes.

What does this page cover?

It gives you a compact reference excerpt plus original lgStudy explanations, examples, applications and study material on His-tag.

Tags

  • Biochemical separation processes
  • Biochemistry detection methods
  • Molecular biology techniques
  • Protein structure

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