His-Tag: Definition & Overview

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Definition: His-Tag

Affinity tags are powerful tools used in life sciences labs for basic biological studies as well as structural and functional proteomics. 

The tags are structurally a short sequence of DNA, coding specific amino acids or proteins. They are inserted into a suitable vector at the coding point of the target gene for its expression and attachment at either C- or N-terminal of the protein. 

There are many types of protein tags used for the purification of specific proteins. One such tag among them is His-tag, also known as polyhistidine tags. His-tags are strings of 6-10 histidine residues.

The histidine tag is predominantly used in protein purification assays to obtain specific fusion proteins. Additionally, it can also enhance the solubility of the proteins of interest during lab assays. Furthermore, the versatility and low immunogenicity of His-tags under denaturing conditions distinguish them from other protein tags.

The molecular weight of the histidine tags varies based on the number of histidine residues involved in making the tag. However, the smallest tag, 6xHis-tag, has a ​molecular weight of 0.8 kDa. 

The size of the His-tag is also a significant factor in purifying recombinant proteins from a mixture of thousands of proteins or cell lysates. The tags of small sizes, such as the 6xHis-tag, have the least possibility of interfering with the target protein functions. However, when using larger tags, such as 10xHis-tag, GST tags, or long peptide tags, the chances of interference increase.

In this article, we will learn about His-tags, their working principle, and diverse applications in life science labs for affinity purification and other assays.

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How To Add His-tag To Proteins?

The small size and charge of His-tags allow them to be easily added to any protein at N- or C-terminus by PCR or site-directed mutagenesis methods.

The code of His-tags is cloned into a suitable plasmid or vector at a chosen location. The gene of interest is subcloned beside the epitope tag with adjacent protease cleavage sites to achieve protein expression and purification. 

You must also note that different expression systems such as E.coli cells or mammalian cells require different cloning approaches. It’s recommended to research a suitable procedure based on your expression system and application. 

His-tag Purification

His-tag protein purification is based on the interaction of the imidazole ring of histidine with divalent metal ions, such as Ni2+ and often Co2+, Cu2+, and Zn2+. The tags bind the metal ions at a pH of 7.5-8. 

You must also know that the length of His-tag also influences its binding with the metal ions. For example, long chains like 10xHis-tag have more binding affinity than smaller tags, such as 6xHis-tags. However, increasing the number of amino acid residues will cause interference in the protein functions.

The technique by which proteins are purified through His tag is immobilized metal affinity chromatography (IMAC). In this method, the divalent ions (or ligands) are immobilized on an agarose/sepharose matrix using chelating agents like nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA).

The His-tagged proteins will strongly bind to the Ni-NTA Agarose resin columns, unlike other proteins that either weakly bind to the matrix or do not bind at all to the column. This helps obtain pure recombinant protein directly from a crude lysate.

To elute His-tagged proteins, low concentrations of imidazole (5-20 mM) followed by high concentrations (100-500 mM imidazole) are used in the process. The low concentration of imidazole reduces nonspecific binding, but a high concentration is used to elute the His-tagged proteins.

Furthermore, a low pH (between 4-5) is also required to elute the His-tagged proteins. However, this might also denature some proteins.

His-tag Applications

Apart from its role in protein purification, the His-tag in proteins also allows proteins’ detection by:

  • Using anti-His-tag antibodies
  • In-gel staining (SDS-PAGE) with fluorescent probes carrying metal ions

The His-tagged fusion protein is also involved in assays like ELISA, western blotting, subcellular localization, and other immuno-analytical workflows.

ELISA

ELISA stands for enzyme-linked immunosorbent assay. It’s an efficient technique used in labs to detect and quantify proteins, peptides, antibodies, and other soluble substances in a complex mixture of thousands of proteins.

Horseradish peroxidase (HRP) chelated with nickel allows the detection of His-tagged proteins without utilizing antibodies or anti-His-tag antibodies.

Western Blot

Western blots are a technique used in labs to detect proteins using specific antibodies after SDS-PAGE experiments. The proteins are transferred on either nitrocellulose or PVDF membrane, incubated with primary antibodies and secondary antibodies, and then visualized using a visualizing reagent.

A range of anti-6xHis-tags is commercially available for their application in western blot assays. 

Pull-down Assay

Pull-down assays are a widely used proteomics method in labs to study protein-protein interactions, verify a predicted protein interaction, and identify the novel interacting partners.

A histidine tag paired with immobilized divalent metal ions is used in the assay to identify and purify a target protein from a complex mixture.

Immunoprecipitation

A method used to precipitate a specific protein antigen out of a solution using specific antibodies. His-tags are used for immune labeling and anti-his-tag antibodies are used in the assay for protein detection and purification. 

Binding Assay

Binding assays work on the principle of the interaction of a ligand with their receptors, such as the binding of a protein with another protein or macromolecules. The technique is used in the same way as pull-down assays. However, it’s less sensitive and can not be used with detergents or in reduced conditions.

The His-tags bound with the metal ions are utilized in the assay to detect and study the protein-protein interactions.

His-tag Industries

Histidine-tags is an essential tool in many life sciences, molecular biology, and biotech labs for proteomics studies. Being a more sensitive tag and its small size, the tags are easily integrated into a spectrum of assays.

In biotech labs, the histidine tag is cloned in a plasmid vector along with the recombinant proteins. It helps in the purification of the tagged proteins. Furthermore, the tags also help in the characterization of the unknown protein.

His-tags are used in a variety of lab workflows including immunoprecipitation, immune labeling, western blot, immunocytochemistry, flow cytometry, and ELISA. The tag facilitates the detection of specific fusion proteins with specificity and their purification. 

Furthermore, the His-tag is also used to study different interactions of protein, such as protein-protein interaction or protein-DNA interaction. 

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Histidine tag or His-tag is an affinity tag used in labs for biological studies. The tags are attached at the N-or C-terminal of the proteins to facilitate protein detection and purification processes.

The His-tag can stretch from 6 histidine residues to 10 histidine residues. However, the small size of the His-tag is extensively preferred in labs because of its less interference with the protein functions. 

The working of His-tag is based on the interaction of the imidazole group that chelates with the divalent metal ions, such as Ni2+ or Co2+. When a protein mixture is introduced into the column, consisting of magnetic beads and sepharose/agarose resin, the tagged proteins are attached to the column and the unwanted proteins are filtered out leaving target proteins.

His-tags have a spectrum of applications in a range of life science and biotech areas, including roles in ELISA, immunoprecipitation, western blot, and flow cytometry assays.

These assays are expensive, and thus, require one to perform them carefully using quality reagents and updated tools and machinery. Doing this will help you not only obtain quality and reliable data but also avoid the repetition of experiments, saving you time and money.

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