The most important application of IMAC is the purification of His-tagged recombinant proteins, and the fusion-expressed His-tag is a fragment containing 6 or more histidine residues. His tag has high affinity and specificity, so in most cases, one-step IMAC purification is sufficient to prepare the target protein with a certain degree of purity, and can meet the requirements of many applications. The structure (i.e., position, order, and length) of tags can affect protein production in several ways: expression rate, binding capacity to IMAC ligands, protein three-dimensional structure, protein crystal formation, etc.
The most common form of His-tag is composed of 6 consecutive histidine residues, which can provide 6 metal binding sites. In most cases, the higher the binding/dissociation equilibrium shift ability, the more stable the binding ability, the more the equilibrium is tilted in the direction of binding. The results of Biacore assay showed that at pH 7.0-7.4, the dissociation rate of hexahistidine-labeled protein from Ni-NTA was 1×10⁶-1.4×10⁸. However, the fluidity, ligand density, and protein concentration of planar chip surfaces are very different from porous agarose particles. Furthermore, the stability of the interaction of His-tagged proteins with IMAC ligands is affected by the accessibility of the tag and the number of total chelating residues (histidine, cysteine, aspartate and glutamate) on the protein surface . That is, usually even under harsh conditions, if the His-tag is accessible (which is the case in most cases), the affinity of the protein for Ni-NTA is high enough for column chromatography.
The most commonly used ligands for IMAC are iminodiacetic acid (IDA) and nitrilotriacetic acid (NTA). coordinating group. Cu2+, Ni2+, Zn2+ these ions exist in the form of six-coordinate compounds in aqueous solution. The chelating ligands have many coordinating groups, which can chelate the metal firmly, but the metal has few sites for protein coordination, and the binding force to the protein is weakened. Therefore, the binding of IDA to protein is generally stronger than that of NTA; however, NTA and metal chelate firmly, and the metal ion is not easy to fall off.
Welch Materials now offers Ni-NTA and Ni-IDA, both packing material and prepacked columns.