- Polyhistidine-tag
A polyhistidine-tag is an
amino acid motif inprotein s that consists of at least sixhistidine ("His") residues, often at the N- or C-terminus of the protein. It is also known as hexa histidine-tag, 6xHis-tag, and by the trademarked name His-tag (registered by EMD Biosciences). The tag was invented by Roche and its vectors are distributed byQiagen . A Variety of purification kits for histidine-tagged proteins are available fromQiagen , Sigma,Thermo Scientific ,GE Healthcare and others.The use of the tag for academic users is unrestricted; however, commercial users must pay
royalties to Roche. Suitable tag sequences are available for free 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. The his-tag may also be followed by a suitableamino acid sequence that facilitates a removal of the polyhistidine-tag usingendopeptidases . This extra sequence is not necessary ifexopeptidases 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.Applications
Protein purification
Polyhistidine-tags are often used for affinity purification of polyhistidine-tagged recombinant proteins that are expressed in "
Escherichia coli " [ [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=7667882&query_hl=4 Trends Biochem Sci. 1995 20(7):285-6] "Purification of His-Tag fusion proteins from Escherichia coli"] or other prokaryotic expression systems. The bacterial cells are harvested bycentrifugation and the resulting cell pellet can be lysed by physical means or with detergents orenzyme s such aslysozyme . The raw lysate contains at this stage the recombinant protein among several other proteins derived from the bacteria and are incubated with affinity media such as NTA-agarose, HisPur resin, or Talon resin. These affinity media contain bound metal ions, eithernickel orcobalt to which the polyhistidine-tag binds with micromolar affinity. The resin is then washed with phosphate buffer to remove proteins that do not specifically interact with the cobalt or nickel ion. The washing efficiency can be improved by the addition of 20 mMimidazole and proteins are then usually eluted with 150-300 mM imidazole, but higher concentrations are used as well. The purity and amount of protein can be assessed bySDS-PAGE andWestern blotting .Affinity purification using a polyhistidine-tag usually results in relatively pure protein when the recombinant protein was expressed in prokaryotic host organism. In special cases or for special purposes like the purification of protein complexes to study protein interactions, purification from higher organisms such as
yeast s or othereukaryote s may require atandem affinity purification [ [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11805826&query_hl=2 Nature. 2002 415(6868):141-7] "Functional organization of the yeast proteome by systematic analysis of protein complexes"] using two tags to yield higher purity. Alternatively, single-step purification using immobilized cobalt ions rather than nickel ions generally yields a substantial increase in purity and requires lower imidazole concentrations for elution of the his-tagged protein.Because its mechanism is only dependent on the primary structure of proteins, polyhistidine-tagging is the option of choice for purifying recombinant proteins in denaturing conditions. Generally for this sort of a technique, histidine binding is titrated using pH instead of imidazole binding -- at a high pH histidine binds to nickel but at low pH (~4) histidine becomes protonated and is competed off of the metal ion. Compare this to antibody purification and GST purification which require some protein be properly folded.
Polyhistidine-tag columns will retain several well known proteins as impurities. One of them is FKBP-type peptidyl prolyl isomerase, which appears around 25kDa (SlyD). These should be separated in any protein prep by using a secondary chromatographic technique, or avoided by expressing the recombinant protein in a SlyD-deficient "E. coli" strain.
Binding assays
Polyhistidine-tagging can be used to detect protein-protein interactions in the same way as a pull-down assay. However, this technique is generally considered to be less sensitive, and also restricted by some of the more finicky aspects of this technique. For example, reducing conditions cannot be used,
EDTA and many types of detergents cannot be used.Adding Polyhistidine Tags
The most common polyhistidine tags are formed of six histidine (6xHis tag) residues which are added at the
C-terminal orN-terminal of theprotein of interest. The choice of the end where His-tag is added will depend mainly on the characteristics of the protein and the methods chosen to remove the tag. Some ends are buried inside the protein core and others are important for the protein function or structure. In those cases the choice is limited to the other end. On the other hand most availableexopeptidases can only remove the His-tag from the N-terminal, therefore removing the tag from the C-terminal will require the use of other techniques.There are two ways to add polyhistidines. The most simple is to insert a protein DNA in a vector encoding a His-tag so that it will be automatically attached to one of its ends .
"Example of primer designed to add a 6xHis-tag using PCR". Eighteen bases coding six histidines are inserted right after the START codon or right before the STOP codon. At least 16 bases specific of a gene of interest are needed next to the His-tag.
Detection
The polyhistidine-tag can also be used in detection of the protein via anti-polyhistidine-tag antibodies in gel staining (
SDS-PAGE ) with fluorescently labelled metal ions. This can be useful in subcellular localization,ELISA ,western blotting or other immuno-analytical methods.Immobilization
The polyhistidine-tag can be ideally used for the immobilization of proteins on a surface such as on a nickel or cobalt coated
microtiter plate or on aprotein array .References
Wikimedia Foundation. 2010.