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Isolating Proteins? Use Histidine and Transition to Metals

Posted in: Protein Expression and Analysis
histidine isolation of proteins

With the advent of recombinant DNA technology, it has now become routine to generate hard-to-isolate proteins by synthetic means. You can clone the desired gene sequence into a vector and generate recombinant proteins. Usually, you use bacterial cells with tailor made genotypes to express your recombinant protein. Efficient production will allow you to perform structural and functional studies.

But the trick in all of this is your ability to isolate pure protein once it has been produced. This is where transition metals can help you out!

Tag Your Protein

To isolate your protein, you are going to need to tag it with something that will allow you to fish it out of the bacterial protein soup. A lot of people opt to introduce a series of histidine residues at either the N-termini or C-termini of their protein sequence, or in some special cases, they decorate both the ends with the histidines. These segments are popularly called His-tags, and are essentially a sequence encoding for six to eight histidines, usually followed by a protease cleavage site.

If you have a good understanding about the histidine clusters occurring naturally in your protein, you may find that you do not need to tag your protein at all!

Why Histidine?

To isolate your tagged protein, you need a matrix onto which the metal is chelated and the bacterial slurry is washed through, in a process termed immobilized metal affinity chromatography (IMAC not iMAC) 1.

Histidine is popular because of its affinity for transition metals and ability to form a transition complex with the tagged-protein. The high selectivity of histidine for transition metals ensures high separation rate and purity.

Metal and Ligand—Both are Required and Make a Difference

The most common metals used with histidine are Ni2+, Co2+, Cu2+ and Zn2+. You can easily purchase pre-packed columns and charge them with the metal of your choice, and voilà, a column ready to purify your desired protein is waiting the next equilibration. Newbie biochemists are unaware of the fact that there is a ligand to which these metals are attached and immobilized on an agarose-based matrix. Iminodiacetic (IDA) and nitrilotriacetic acid (NTA) are the two commonly employed ligands and do play a role in the yield and purity of the recombinant proteins.

Although Ni2+ is the most commonly used metal for IMAC, it is always worth testing the other metals to check the yield and purity of your desired proteins. The results can be rather surprising and useful. Therefore, a combination of the ligand and metal can influence the sample quality, especially if you do not intend to remove the His-tag.


Like any other technique, IMAC is not flawless. Native histidines in the protein can also bind to the metals and you can be left with contamination from undesired proteins (common E. coli proteins are metalloproteins and chaperones 2). You also have to watch out for metal leaching out from the column, sequestered by the His-tags. This is especially important when crystallizing a protein as usually very little is done to ensure a metal-free protein sample prior to crystallization. And always remember, some enzymes are inactivated in presence of metals! In such troublesome cases, variation in the imidazole concentration, tinkering with pH and salt helps. Co-tagging, using more than one type of tag, also is a useful strategy.

So the next time you glance at the periodic table, take special notice of those unique transition metals sitting in groups 8-12. They are friendlier to a biochemist than you may think.


  1. Sundberg, R. J.; Martin, R. B. 1974. Interactions of histidine and other imidazole derivatives with transition metal ions in chemical and biological systems. Chem Rev. 74(4): 471-517.
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  2. Bolanos-Garcia, V.; Davies, O. 2006. Structural analysis and classification of native proteins from E. coli commonly co-purified by immobilised metal affinity chromatography. Biochimica et Biophysica Acta (BBA)-General Subjects. 760, (9):1304-1313.
    (Back to article)


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