Polyhistidine-tag

A polyhistidine-tag is an amino acid motif in proteins that typically consists of at least six histidine (His) residues, often at the N- or C-terminus of the protein. It is also known as hexa histidine-tag, 6xHis-tag, His6 tag, by the US trademarked name HIS TAG (US Trademark serial number 74242707), and most commonly as His-Tag. The tag was invented by Roche, although the use of histidines and its vectors are distributed by Qiagen. Various purification kits for histidine-tagged proteins are available from Qiagen, Sigma, Thermo Scientific, GE Healthcare, Macherey-Nagel, Cube Biotech, Clontech, Bio-Rad, and others. A polyhistidine-tag is an amino acid motif in proteins that typically consists of at least six histidine (His) residues, often at the N- or C-terminus of the protein. It is also known as hexa histidine-tag, 6xHis-tag, His6 tag, by the US trademarked name HIS TAG (US Trademark serial number 74242707), and most commonly as His-Tag. The tag was invented by Roche, although the use of histidines and its vectors are distributed by Qiagen. Various purification kits for histidine-tagged proteins are available from Qiagen, Sigma, Thermo Scientific, GE Healthcare, Macherey-Nagel, Cube Biotech, Clontech, Bio-Rad, and others. The use of the tag for academic users was unrestricted; however, commercial users had to pay royalties to Roche. The original patent expired on 11 Feb 2003, and is now public property; current claims to royalties are based on a much narrower set of more recent patents. Suitable tag sequences are available free for commercial use; for example, MK(HQ)6 may be used for enhanced expression in E. coli and tag removal. The total number of histidine residues may vary in the tag from as low as two, to as high as 10 or more His residues. N- or C-terminal his-tags may also be followed or preceded, respectively, by a suitable amino acid sequence that facilitates a removal of the polyhistidine-tag using endopeptidases. This extra sequence is not necessary if exopeptidases are used to remove N-terminal His-tags (e.g., Qiagen TAGZyme). Furthermore, exopeptidase cleavage may solve the unspecific cleavage observed when using endoprotease-based tag removal. Polyhistidine-tags are often used for affinity purification of genetically modified proteins. In general, proteins possess more or less the ability to coordinate metal ions on their surface, and it is possible to separate proteins by chromatography making use of the difference in their affinity. This is the immobilized metal ion affinity chromatography announced in 1975. Subsequent studies have revealed that among amino acids constituting proteins, histidine is strongly involved in the coordinate bond with metal ions. Therefore, if a number of histidines are added to the end of the protein by genetic engineering, the affinity of the protein for the metal ion is remarkably increased and the basic idea is that purification can be easily carried out. When a protein having a His tag is brought into contact with a carrier on which a metal ion such as nickel is immobilized under the condition of pH 8 or higher, the histidine residue chelates the metal ion and binds to the carrier. Since other proteins do not bind to the carrier or bind only very weakly, it can be removed by washing the carrier with an appropriate buffer. Thereafter, by removing imidazole or the like from the carrier, it is possible to recover the protein having the His tag with high purity. Various carriers such as Ni - NTA agarose (nickel - nitrilotriacetic acid) are on the market. It is packed in a column and used in combination with centrifugation and magnetic separation in a test tube. As the metal ion, copper has the highest affinity, and the affinity decreases in the order of nickel, zinc, and cobalt. Nickel is often used for ordinary purposes, and cobalt is used when it is desired to increase the purity of purification.