The HIS-story of Protein Tags

If you plan on producing a recombinant protein these days, you are spoilt for choice of tags. 

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The HIS-story of Protein Tags

Recombinant proteins began with recombinant DNA (thank you captain obvious!). When researching back into the recesses of history, I found the first mentions of recombinant DNA in a paper in 19721 talking about using restriction endonucleases to create staggered (sticky) ends of DNA and how these could be ‘stuck’ to DNA from other species.  

Once the trick of taking a gene from one organism and sticking into an expression vector that was then made in a different organism was mastered, this foreign protein now had to be purified.

Chromatography is defined in the Merriam Webster dictionary as “a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the solutes as they flow around or over a stationary liquid or solid phase.”

Tags have been used for years to purify recombinant proteins, as well as being used to identify them when an antibody is unavailable.

Let’s talk about the His-tory of the His Tag. The His TagTM consists of 6-9 Histidine residues and was invented by Roche2, luckily the original patent for commercial use of this tag expired on 11 Feb 2003. This Tag can be cleaved by some available peptidases including Thrombin, TEV Protease and Enterokinase. The principle of this technique is the coordination between the electron donor groups on a protein (peptide) surface and immobilized transition metal ions.

The His- tag can be used in immobilised metal affinity chromatography (IMAC) as it binds to metal ions such as Cu2+ > Ni2+ > Zn2+ in order of highest to lowest affinity. Zinc loading is most widely used to select polypeptides with a hexa-histidine tag, whereas copper is used to select species with one to three histidine residues3.IMAC involves immobilising metal ions through chelation to a matrix and passing your sample over this matrix. Although copper resins bind to His-Tags with the highest affinity, it is the least specific of the metal ions and is therefore usually used for applications where purification is not the primary goal.

We use a Nickle Resin as it has higher binding affinity than Zinc but is more specific and proteins can be eluted by reducing the pH or using a chelating agent such as ethylenediaminetetraacetic acid (EDTA)3 or a strong concentration of Imidazole. Imidazole competes with the his-tag for binding to the metal-charged resin and thus is used for elution as well as a low concentration added to both binding and wash buffers to interfere with the weak binding of other proteins and to elute any proteins that weakly bind.

Another advantage of Tags is that if you don’t have an antibody available towards your protein of interest, you can easily detect the tag with an anti-Tag antibody such as an Anti-His or Anti-GST.

Stay tuned for the next chapter of “The HIS-story of Protein Tags” where we’ll take a look at the history of the GST Tag.


  1. Mertz, J. E. & Davis, R. W. Cleavage of DNA by R 1 restriction endonuclease generates cohesive ends. Proc. Natl. Acad. Sci. U. S. A. 69, 3370–3374 (1972).
  2. Hochuli, E., Bannwarth, W., Dobeli, H., Gentz, R. & Stuber, D. Genetic Approach To Facilitate Purification of Recombinant Proteins With a Novel Metal Chelate Adsorbent. Nat. Biotechnol. 1321–1325 (1988).
  3. Antibody, M., Chromatography, A., Sequence, A. T., Sequence, C. T. & Otter, D. Immobilized Metal Affinity Chro- matography PROTEIN | Determination and Charac- terization Phosphoproteomics Affinity Targeting Schemes for Bio- marker Research. (2003).



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