What is Ion Exchange Chromatography Used For? 4 Important Applications for Protein Purification

Ion Exchange Chromatography is routinely sandwiched between affinity and size exclusion steps in an overall protein purification protocol. However, ion exchange is not just some forgotten middle child in the protein purification family tree. Just the opposite: ion exchange has unique strengths that are leveraged for unique applications.

Ion exchange chromatography (IEX) is used to separate proteins based on their net charge. Four specific uses for IEX are: purifying native proteins, purifying proteins with different posttranslational modifications, separating distinct protein conformations, and isolating discrete oligomeric states.

• Purification of native proteins
• Purification of different posttranslational modification states
• Separation of distinct protein conformations
• Isolating discrete oligomeric states

In related articles, we cover ion exchange chromatography in general, how to pick an ion exchange purification protocol, and describe different ion exchange chromatography resins. In this article we’ll discuss how ion exchange is uniquely suited for these specific purification applications.

Article Table of Contents:

Native Protein Purification

Posttranslational Modifications

Conformational Changes

Oligomeric State

Related Products

References

Native Protein Purification

Genetic engineering is used all the time nowadays to append affinity tags onto different proteins of interest. In general, this is a really powerful strategy, but sometimes this is not a desirable route for a number of reasons.

For one, you might want to purify the protein from its native context to retain relevant posttranslational modifications or binding partners. Alternatively, you may have tried adding an affinity tag and found that it interfered with an important feature of your protein of interest – like its activity or binding partners.

In these cases, you can use the classic method of purifying your native tag-less protein of interest using ion exchange chromatography, or a series of ion exchange steps.

Since a subset of cellular proteins will have similar charge properties to your protein of interest, it is often desirable to use multiple ion exchange purifications with different resins to increase the purity of your protein of interest. For example, both cation exchange and anion exchange purification protocols are used to purify Xrn1, an RNA exonuclease (Pellegrini et al, 2008).

In general, using two different ion exchange steps in tandem helps eliminate contaminating proteins compared to a single purification step (Figure 1). However, when employed downstream of affinity chromatography, a single ion exchange purification step usually produces satisfactory purity.

Figure 1. Multiple IEX purification steps can enhance protein purity, as shown on a hypothetical SDS-PAGE gel. When purifying your protein of interest (green band), most contaminating proteins (blue bands) will get purified away using either anion (AEX) or cation exchange chromatography (CEX). However, a few contaminating proteins may coelute with your protein – here represented as orange and purple bands for AEX and CEX, respectively. By running an AEX purification then a CEX purification you can get rid of both of these contaminants and have more purity for your protein of interest.

Posttranslational Modifications

Ion exchange chromatography is an ideal method for separating a protein that has posttranslational modifications from one that doesn’t. As a reminder, posttranslational modifications are chemical changes that modify amino acids after the protein has been translated.

Now ion exchange only separates posttranslational modifications that change the charge of a protein, such as phosphorylation and acetylation (Figure 2). The resulting mass change from posttranslational modifications is too small to resolve by size-exclusion chromatography and will be purified the same as an unmodified protein by affinity tag purification. But the change in charge can be leveraged to separate the proteins by ion exchange.

Figure 2. Phosphorylation of tyrosine residues and acetylation of lysine residues are dynamic and reversible modifications that change the charge on their side chains.

For example, using a high-resolution resin and a shallow enough gradient, ion exchange can separate different phosphorylation states. Researchers studying a protein that could be phosphorylated up to five times were able to resolve the unphosphorylated, mono-, di-, tri-, tetra-, and penta-phosphorylation states using a MonoQ column and a shallow gradient (Figure 3)(Desjardins et al, 2014; Pufall et al, 2005).

Figure 3. High resolution ion exchange columns and a shallow elution gradient are used to separate unphosphorylated from mono-, di-, tri-, tetra-, and pentaphosphorylated protein (Desjardins et al, 2014; Pufall et al, 2005). Protein is represented by the circle and squiggle, and phosphorylation modifications are represented by “P”.

Conformational Changes

Ion exchange chromatography is very useful in separating different protein conformations. First, we need to talk a little about what a protein conformation is.

When we look at images of proteins, whether they are actual structures or just representative renderings, it gives us the impression that proteins are static objects like a rock. In reality, proteins are incredibly dynamic and interconvert between different conformations.

These conformations are often subtle. Consider Figure 4 – can you spot the differences between these two protein conformations? I’ll give you a minute to scrutinize the two structures …

They’re the same picture, right? No, just kidding! If you look closely, you’ll see the appearance of an additional a-helix in the top right corner of the right conformation that doesn’t exist in the left conformation.

You may have also noticed that the a-helices at the bottom of the structure are more tightly packed together in the left conformation.

And actually, one of the most crucial changes might also be the most subtle. This change involves the bundle of a-helices that run lengthwise from top to bottom of the protein. In the left conformation they are splayed open at the top and tight together on the bottom. The right conformation is just the opposite with the helices spread apart at the bottom and bundled together at the top (Nomura et al, 2015). This change is actually really important for the protein’s function – check out Figure 4’s legend for more details on that.

Figure 4. Glut5, a mammalian sugar transporter, shifts between two slightly different conformations (Nomura et al, 2015). Glut5 sits in the membrane, and these proteins are oriented such that the top would be outside of the cell and the bottom would be inside the cell. Glut5 on the left is in an “outside open” conformation, where the a-helices at the top of the protein are splayed apart to let sugar molecules from outside the cell enter the protein. To let those sugar molecules into the cell, the protein must shift to an “inside open” conformation (right), where the a-helices at the bottom of the protein are now splayed apart.

If you are having trouble seeing the changes in Figure 4 – don’t worry, that is kind of the point: the overall protein fold is still very similar, and conformational changes are often fairly subtle.

However, conformational changes can also be quite dramatic – such as the entire protein rearranging to take on an entirely different structure. For example, a domain in the bacterial protein RfaH changes into a completely different shape (Figure 5) (Belogurov et al, 2007; Burmann et al, 2012; Mooney et al, 2009). This structural change is important because in the a-helical state (Figure 5, left), the protein regulates transcription, but in the b-barrel state (Figure 5, right), the protein regulates translation.

Getting back to ion exchange chromatography, different protein conformations may have distinct surface charges because a unique subset of (charged) amino acids will be exposed to solvent and available for binding to ion exchange resin. In a subtle case, like what is shown in Figure 4, it may be that the changes are so miniscule that it is impossible to separate the conformations by ion exchange. However, in a more dramatic case, like Figure 5, researchers separate different conformations with a shallow gradient elution and a high-resolution resin.

Figure 5. RfaH C-terminal domain (CTD) shapeshifts between two different conformations. In the a-helical conformation (left), RfaH regulates transcription whereas in the b-barrel conformation (right), RfaH regulates translation (Belogurov et al., 2007; Burmann et al, 2012; Mooney et al, 2009).

Oligomeric State

Another useful application of ion exchange chromatography is separating the same protein based on its oligomeric state. Most commonly this means separating the functional state, which is usually a monomer or a dimer, from an aggregate or a very large assembly state. The larger the assembly state, the more charged the protein assembly will be, and therefore the later it will elute off of the column if you are doing a gradient elution (Figure 6). Think of it this way – if your protein has an overall net charge of +3, then a decamer – an oligomer having 10 copies of the protein – will have an overall net charge of +30 (+3 x 10).

Some proteins have discrete functional assemblies, and for these proteins performing a high-resolution ion exchange purification can separate these distinct assemblies from one another. For example, ion exchange was used to separate dimer, tetramer, hexamer, and octamer assemblies of bovine serum albumin (Amartely et al, 2018).

Figure 6 . When a protein has different oligomeric assemblies, the lower subunit assemblies, such as a monomer, will elute at lower salt concentrations and the higher subunit assemblies will elute at higher salt concentrations. Dashed line represents the increasing salt concentration during the purification.

You probably noticed that throughout this article we’ve mentioned the importance of shallow gradient elutions in these applications. We have a great article that takes a deep dive into ion exchange purification protocols and gradient elutions – check it out for more information on this topic.

So hopefully now you can appreciate the power of ion exchange chromatography. If you are purifying a native protein, separating posttranslational modifications, a specific protein conformation, or different oligomeric assemblies, ion exchange is a great purification choice!

 

References

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