A polyhistidine tag, commonly known as a His tag, is a short, genetically engineered sequence of amino acids used as a molecular handle for a target protein. This tag is typically comprised of six to ten consecutive histidine residues, with the hexa-histidine (6xHis) version being the most frequently employed. The N-terminal designation indicates that this sequence is added to the very beginning of the protein’s amino acid chain. This small addition does not fundamentally change the protein’s overall structure or function. Instead, its sole purpose is to provide a specific chemical characteristic that allows researchers to isolate the recombinant protein from a complex mixture of thousands of other cellular proteins.
Why Polyhistidine Tags Are Essential
The utility of the His tag stems from its highly specific chemical affinity for certain transition metal ions. This feature makes it a rapid and efficient purification tool in biochemistry. The tag’s small size minimizes the risk of interfering with the target protein’s folding, structure, or inherent biological activity.
Using the His tag enables researchers to achieve a high degree of protein purity, often reaching 95% in a single step, with an enrichment factor of up to 100-fold. This speed and yield surpass many other purification methods, making it the preferred choice for initial protein isolation. Beyond purification, the His tag has secondary applications, such as acting as an epitope that can be recognized by commercially available anti-His antibodies. This allows the tagged protein to be easily detected and quantified in experiments like Western blotting, confirming its presence and concentration within a sample.
How Immobilized Metal Affinity Chromatography Works
The His tag’s function is realized through Immobilized Metal Affinity Chromatography (IMAC), which exploits the tag’s unique metal-binding properties. This method relies on a chromatography column packed with a porous resin, which serves as the solid matrix. Chelation agents, such as nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA), are chemically bonded to this resin, acting as anchors for divalent transition metal ions like Nickel (\(text{Ni}^{2+}\)) or Cobalt (\(text{Co}^{2+}\)). Nickel is the most common choice due to its high affinity and relatively low cost.
When the crude cell lysate containing the target protein is passed through the IMAC column, the histidine residues in the tag bind to the immobilized metal ions. Each histidine side chain contains an imidazole ring, a nitrogen-containing structure that readily forms coordination bonds with the \(text{Ni}^{2+}\) or \(text{Co}^{2+}\) ions attached to the column matrix. This forms a stable, reversible complex, causing the His-tagged protein to stick tightly to the column while all other untagged cellular proteins are washed away.
The bound protein is then separated from the column in a controlled process called elution. The most common elution method involves passing a buffer containing a high concentration of free imidazole through the column. Imidazole mimics the side chain of histidine and acts as a competitive agent. Because of its high concentration (often 250 to 500 millimolar), the free imidazole molecules flood the column and outcompete the His tag for the binding sites on the metal ions, releasing the target protein in a highly purified form.
Designing the Tag: Placement and Cleavage
Integrating the His tag requires careful engineering decisions regarding its placement and the necessity of its eventual removal. The choice between placing the tag at the N-terminus (the start) or the C-terminus (the end) is highly protein-specific. N-terminal placement is often favored because it is generally more accessible for binding to the IMAC column. However, in some cases, the placement can interfere with the protein’s native folding or biological activity.
Although the His tag is small, it must often be removed after purification if the protein is intended for sensitive downstream applications like X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. The presence of the tag can sometimes cause the protein to aggregate or interfere with its native structure and function. To enable this removal, a specific amino acid sequence, known as a protease cleavage site, is engineered between the His tag and the target protein sequence.
A common example is the cleavage site for TEV protease, which recognizes the sequence Glu-Asn-Leu-Tyr-Phe-Gln↓Gly and precisely cuts the peptide bond at the specified location. After the initial IMAC purification, the purified, tagged protein is incubated with the specific protease, which clips the tag off the protein. A final, secondary purification step is then performed by passing the cleaved mixture back over a fresh IMAC column. The detached His tag, any uncleaved protein, and often the engineered protease itself will rebind to the column, while the newly liberated, tag-free protein flows through and is collected.