What Is a His-Tag?
by Simon Currie
A His-tag is a stretch of 6-10 histidine amino acids in a row that is used for affinity purification, protein detection, and biochemical assays. His-tags bind to metal ions such as nickel or cobalt, and this interaction is how his-tagged proteins are purified with metal-conjugated agarose beads.
His-tags are popular affinity tags that are used for protein purification, detection, and many different assays. But if you’re new to the protein biochemistry space, you might be wondering what is a “his-tag” is. If so, this is a great article for you.
A His-tag is a stretch of 6-10 histidine amino acids in a row that is used for affinity purification, protein detection, and biochemical assays. His-tags bind to metal ions such as nickel or cobalt, and this interaction is how his-tagged proteins are purified with metal-conjugated agarose beads.
Regardless of how long the his-tag is, it will still do the same thing: bind to metal ions. The longer the tag, however, the tighter the interaction. So longer his-tags can provide better purity in protein purification, and better sensitivity in detection and binding assays.
The his-tag isn’t always retained on the protein after purification, but is sometimes cut off and purified away during the purification itself.
In this article, we’ll compare his-tags with other affinity tags, discuss how his-tags bind to metal and how that interaction is used for protein purification and in different assays.
Article Table of Contents
His-tag interaction with nickel ions
How long should my his-tag be?
Optimizing his-tag purification with imidazole
His-tags vs other affinity tag options
Why use a his-tag?
Before genetic cloning and recombinant technologies were developed, proteins used to be purified from sources such as animals’ organs. You would head down to the local butcher, get all of the cow livers or calf thymuses that they had in stock, and hurry back to lab to grind up these organs and purify your protein based on its charge and size.
Imagining this is pretty brutal. And it wasn’t fun for the scientists either. Thankfully, we usually don’t need to do that anymore.
Nowadays, most protein purification comes after cloning a plasmid to express your protein recombinantly in an organism like Escherichia coli. This removes the time-consuming and unpleasant process of acquiring animal organs and grinding them into pulp. It also makes the protein purification more reproducible and scalable compared to the old way of doing things.
When you’re cloning your plasmid, there is an opportunity to add a “tag” to your protein of interest. By “tag,” I mean some amino acid sequences that are not from the protein of interest itself. A common addition is to add an affinity tag like a his-tag for quick and easy purification of the protein of interest.
A his-tag is a commonly used affinity tag, and its interaction with nickel and cobalt beads is used to purify his-tagged proteins.
One way to understand this is to think about luggage tags. Imagine you’re in a big family, and you all have pretty similar suitcases. As you wait at baggage claim in the airport for your suitcase, you see a lot of similar suitcases going around the carousel. The way to easily identify yours from anyone else’s is the luggage tag.
Similarly, a his-tag, or any affinity tag, is like a combination between a luggage handle and the luggage tag on your suitcase at the airport. The his-tag helps you both identify and pull your protein of interest out away from a sea of other proteins.
His-tag interaction with nickel ions
The side chain of histidine residues binds to nickel ions. By stringing histidine residues together, you form a “his-tag” that will bind to nickel agarose beads while most other proteins flow through.
Histidine residues are found in most proteins. In some proteins, a group of histidine residues will be sufficiently close in the protein’s primary sequence, or in its 3D structure, to act like a naturally occurring his-tag (Hemdan et al, 1989; Salichs et al, 2009). Host proteins with such histidine-rich sequences will bind to the nickel agarose beads, in addition to the his-tagged protein that you are trying to isolate (Figure 1).
Figure 1. Contaminating host proteins (orange and pink proteins) have histidine residues and also bind to the nickel agarose beads in the absence of imidazole.
The length of your protein’s his-tag and the concentration of imidazole that you include in your load and wash buffer is fine-tuned to facilitate binding of your protein to the column while washing all other contaminating proteins away. We’ll discuss this concept more in the next section, as well as in this article.
How long should my his-tag be?
Typically, his-tags are between 6 and 10 histidine residues in length. The longer the his-tag, the tighter it will bind to nickel beads.
The difference in binding affinity to nickel columns is due to avidity, meaning that there are more histidine residues that can bind to the nickel ion.
Let’s imagine you are cleaning your house or apartment. Since you only have 2 arms to pick up and clean, it takes you a certain amount of time. But if you had 4 arms to work with, you would be able to clean much faster. Similarly, more histidine residues in your his-tag is like having more arms (or hands) for the his-tag to grab onto the nickel agarose beads with (Figure 2).
Figure 2. Shorter his-tags are like having less hands to grab onto the nickel agarose beads. Some shorter his-tags will still bind, but many will miss (left). A longer his-tag increases the interaction strength between the his-tag and the nickel ion meaning that a higher percentage of the his-tagged proteins will grab onto the nickel (right).
Longer his-tags binding tighter to nickel columns means that you can also get more pure protein if you design your purification smartly.
Optimizing his-tag purification with imidazole
Imidazole is used to elute his-tagged proteins from nickel beads. Including a lower concentration (~ 5 to 30 mM) of imidazole in your binding and wash buffers also reduces non-specific binding from other proteins to the nickel column (Figure 3).
Figure 3. Including a low concentration of imidazole (I) in the wash buffer prevents contaminating host proteins from binding to the nickel agarose beads.
How important is this difference in purity? Well, it depends on if you’re doing any downstream purification steps, and what application you’re using the protein for. If you plan on performing additional purification steps, such as ion exchange or size exclusion chromatography, then a small difference in purity after the nickel elution step likely won’t matter by the end of your purification. However, if you’re only doing the nickel affinity purification step then you’ll want to maximize your protein’s purity.
His-tags vs other affinity tag options
His-tags are not the only kind of affinity tags. Other short peptide tags include strep tags and flag tags, which we talk about in this article. Additionally, if your protein of interest is aggregation-prone or expresses poorly, you might want to use a larger solubility tag such as GST, MBP, or SUMO tags, which we cover here.
Each of these tags has an ideal use scenario, a context in which they are the superior choice relative to the others. However, his-tags are the most commonly used affinity tag. There are three main reasons why his-tags are used so frequently:
- - His-tags are small and usually do not interfere with a protein’s structure or function
- - The reagents for purifying his-tagged proteins are less expensive
- - His-tags are useful in a range of different experimental applications
The small size of his-tags means that you can usually add them to the N- or C-terminus of proteins, and it usually will not interfere with that proteins’ structure, function, or interaction with other binding partners.
If a his-tag does interfere with your protein’s function, it’s possible to keep the his-tag throughout purification, then cut it off afterwards.
To do so, you’ll need to include a protease cleavage site between the his-tag and the protein, which can be added during the cloning stage.
For example, if you’re purifying an enzyme and the his-tag disrupts the enzyme’s function by blocking the active site, then you can cleave the his-tag off to restore enzyme activity. (Figure 4).
Figure 4 The his-tag blocks the active site of the enzyme (left), but after a protease cleaves off the his-tag the enzyme can bind to its substrate again (right).
In this way, including the protease cut-site gives you versatility in deciding if you want to keep the his-tag, or not, for a given purification or purpose.
Relative to other affinity tags, nickel agarose beads and other reagents used to purify his-tags are less expensive and easy to use, especially when you consider that you can clean and regenerate nickel agarose beads with just some nickel and basic lab buffers. The savings really start to add up.
His-tag applications
There are additional research applications for his-tags beyond protein purification. If you’re purifying a protein for use in any of these applications, then you would certainly want to keep the his-tag on the protein and not cleave it off.
- Detection: you can detect the his-tag on your protein in assays such as ELISA, Western blotting, and microscopy.
- Fluorescent tagging: you can bind fluorophores to the his-tag, enabling fluorescent microscopy and other fluorophore-based techniques (Zhao et al, 2010).
- Binding assays: his-tags are used in multiple assays to investigate molecular interactions:
o His-tag pull-down: similar to GST pull-downs, but you’re using the his-tag for isolation or to detect interactions instead.
o Surface Plasmon Resonance (SPR): the his-tag can be used to immobilize the protein of interest for SPR.
o Förster resonance energy transfer (FRET): an energy transfer assay where the his-tag is used to bind to either the donor or acceptor bead.
So, now that you know what a his-tag is, it may be time to start using one for your protein purification needs. Check out below for high-quality products and resources that can help you get going in the lab.
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