Polyhistidine is a synthetic amino acid polymer created from multiple, repeating units of the amino acid histidine. Amino acids are the fundamental building blocks that link together in long chains to form proteins. The defining feature of histidine is its side chain, an imidazole ring, which possesses unique chemical properties.
The Polyhistidine-Tag in Protein Engineering
The most common application of polyhistidine is the polyhistidine-tag, or His-tag, a tool used in protein engineering. Scientists use recombinant DNA technology to genetically fuse a sequence encoding six to ten histidine residues to the gene of a target protein. This tag can be attached to either the beginning (N-terminus) or the end (C-terminus) of the protein’s genetic blueprint.
Once this engineered gene is inserted into a host organism, such as bacteria or yeast, the cellular machinery reads the modified genetic code. This process results in the production of the desired protein with the His-tag physically attached as an extension of its amino acid chain. The small size of the tag rarely interferes with the protein’s normal folding or function. This process yields a large quantity of the target protein, now tagged for isolation from the other native proteins in the cell.
Purification Using Metal Affinity Chromatography
The His-tag enables a highly specific purification method called Immobilized Metal Affinity Chromatography (IMAC). This technique relies on the strong and reversible interaction between the histidine residues and certain metal ions. The process uses a column packed with a porous resin (the stationary phase). These resin beads are chemically coated with chelating agents, such as nitrilotriacetic acid (NTA), that immobilize positively charged metal ions, such as nickel (Ni²⁺) or cobalt (Co²⁺).
When a crude mixture of cellular contents, known as a lysate, is passed through the column, the imidazole ring on the side chain of each histidine in the His-tag forms a coordinate bond with the immobilized metal ions. Because of this strong interaction, only His-tagged proteins adhere to the resin while all other untagged proteins flow through the column and are washed away.
Protein Recovery and Tag Removal
After the untagged proteins have been washed away, the purified His-tagged protein is recovered from the column through a process called elution. A solution containing a high concentration of a competing molecule, free imidazole, is passed through the column. The structure of imidazole is identical to the side chain of histidine, and at high concentrations, it outcompetes the His-tag for binding to the metal ions on the resin.
This competition displaces the tagged protein from the resin, allowing it to be collected in a purified form. Once isolated, the His-tag may need to be removed if its presence could affect the protein’s structure or function in subsequent experiments. Scientists can accomplish this by including a specific recognition sequence for a protease, an enzyme that cuts protein chains, in the linker region between the protein and the tag. Adding this protease cleaves the tag, which is then separated from the final protein product.