How Does His-Tag Purification Work?
A his-tag, or polyhistidine tag, is a string of histidine residues at either the N or C terminus of a recombinant protein. There can be from four to ten residues in a string, although commonly there are six histidine residues — a hexahistidine tag. Some recombinant proteins are engineered to have two hexahistidine tags.
His-tag purification uses the purification technique of immobilized metal affinity chromatography, or IMAC. In this technique, transition metal ions are immobilized on a resin matrix using a chelating agent such as iminodiacetic acid. The most common ion for his-tag purification of a recombinant protein is Ni2+, though Co2+, Cu2+, and Zn2+ are also used. The his-tag has a high affinity for these metal ions and binds strongly to the IMAC column. Most other proteins in the lysate will not bind to the resin, or bind only weakly. The use of a his-tag and IMAC can often provide relatively pure recombinant protein directly from a crude lysate.
Imidazole competes with the his-tag for binding to the metal-charged resin and thus is used for elution of the protein from an IMAC column. Typically, a low concentration of imidazole is added to both binding and wash buffers to interfere with the weak binding of other proteins and to elute any proteins that weakly bind. His-tagged protein is then eluted with a higher concentration of imidazole.
Ni2+ is most commonly used for his-tag purification since it gives a high yield. Using Co2+ can give higher purity but with a lower yield. Bio-Rad has his-tag resins and his-tag purification kits that are precharged with Ni2+ for fast, easy his-tag protein purification. Uncharged resins and kits that can be charged with Co2+ or other divalent or trivalent metal ions are also available. Uncharged kits give users the option of trying different metals to determine if one gives higher purity or yield for a particular his-tagged recombinant protein.
His-tag Purification Features
For his-tag purification, a longer his-tag or two hexahistidine tags provides a stronger affinity for the matrix. As the number of histidine residues increases, the elution buffer requires a higher concentration of imidazole to remove bound proteins from the column. Stronger binding can be an advantage when the crude protein mixture contains unwanted proteins that have a high affinity for the charged IMAC resin. Most nonrecombinant proteins bind more weakly than a long string of histidines and so elute at a lower concentration of imidazole.
Stronger binding to an IMAC column can be particularly useful when the recombinant his-tagged proteins are from a eukaryotic source. Generally, there are fewer proteins with strong affinity for transitional metals in lysates from prokaryotes than from eukaryotes. The use of two hexahistidine tags combined with Co2+ (or Zn2+) can reduce the binding affinity for many of these proteins relative to the his-tagged protein.
If imidazole is not suitable for elution, then the pH of the his-tag column can be lowered to elute the recombinant protein. Lowering the pH causes the metal ions to become protonated, and then they are stripped from the column. All Bio-Rad his-tag resins are stable from pH 1–14, and can be utilized with any protocol that involves changing pH.
When preparing crude lysates for his-tagged protein purification, often one or more detergent or denaturing or reducing agent must be included in the lysis buffer. All Bio-Rad his-tag protein purification kits are compatible with all common types of denaturing and reducing agents and detergents. There is no effect on binding to or elution from the IMAC resin in the presence of these compounds.
His-Tag Purification Benefits
The Bio-Rad range of his-tag purification kits, columns, and media are optimized for high levels of purification at fast flow rates. Due to the very low levels of metal ion leakage from our affinity media, there is no problem with metal ion contamination of samples when using a Bio-Rad his-tag purification kit or column. All resins are stable at low pH, compatible with detergents and both denaturing and reducing agents, have high mechanical strength, and can be used at fast flow rates.