Polyhistidine-tag

Polyhistidine-tag or His-tag is a protein tag that consists of multiple histidine (His) residues, typically six to ten, which is added to the N-terminus or C-terminus of a protein. This tag is widely used in protein purification and protein interaction studies due to the affinity of the histidine residues for certain metal ions, such as nickel (Ni²⁺), cobalt (Co²⁺), and copper (Cu²⁺).

Overview

The polyhistidine-tag is a popular tool in molecular biology and biochemistry for the isolation and purification of recombinant proteins. The technique exploits the affinity of the histidine residues for immobilized metal ions, a principle that forms the basis of immobilized metal affinity chromatography (IMAC). Proteins tagged with a polyhistidine sequence can be selectively bound to a column containing immobilized metal ions and later eluted by increasing the concentration of imidazole in the elution buffer or by decreasing the pH, which competes with the histidine residues for binding to the metal ions.

Applications

Polyhistidine-tags are used in various applications, including:

• Protein Purification: The primary use of His-tags is to facilitate the purification of recombinant proteins. The tag allows for a single purification step that can yield highly pure protein.
• Protein Interaction Studies: His-tagged proteins can be immobilized on a surface and used to study protein-protein or protein-DNA interactions.
• Structural Biology: His-tagged proteins are often used in X-ray crystallography and NMR spectroscopy for structure determination.
• Therapeutic Proteins: Some therapeutic proteins are produced as His-tagged versions to simplify purification, although the tag is usually removed before the protein is used therapeutically.

Advantages and Disadvantages

Advantages

• Simplicity: The use of His-tags simplifies the protein purification process, often allowing for a single chromatographic step.
• Versatility: His-tags can be added to either end of the protein and can be used with a wide range of proteins.
• High Purity: Proteins can be purified to high levels of purity using IMAC.

Disadvantages

• Potential Impact on Protein Function: The addition of a His-tag may affect the structure and function of the purified protein, although this can often be mitigated by removing the tag after purification.
• Metal Ion Leaching: Metal ions can leach from the column and contaminate the purified protein, which may be problematic for certain applications.

Removal of His-tags

In cases where the His-tag may interfere with the protein's function or where the tag needs to be removed for downstream applications, several strategies can be employed. These include the use of protease cleavage sites engineered between the His-tag and the protein, allowing for the tag's removal by specific proteolytic cleavage.

See Also

• Protein purification
• Recombinant DNA
• Immobilized metal affinity chromatography
• Protein tag