Simple Overview of the 4 Common Techniques for Protein Purification

Figure 1. Target proteins with an affinity tag bind to agarose beads with a ligand (2^nd column) and are then eluted (3^rd column).

However, affinity purification wasn’t always a tool in scientists’ toolkits for purifying proteins.

Historically, proteins were purified from tissues, organs, and microorganisms such as red blood cells (hemoglobin), cow pancreas (insulin), and the pathogenic bacteria Clostridium botulinum (Botox^TM). In their native form, these proteins lack affinity tags that can streamline the purification process.

The advent of modern molecular biology meant scientists are no longer stuck just working with proteins from natural sources. We can now modify the DNA that encodes for a protein to create custom proteins with desired features and express these customized proteins in a variety of host organisms for downstream purification.

These proteins are called recombinant proteins since they are expressed from recombinant (re-combined) DNA.

When designing a custom recombinant protein, scientists often add an affinity tag to aid in protein purification or target protein detection.

Think about an affinity tag like the handle on a pot – you don’t necessarily have to have a handle to cook food on the stove, but it makes handling the pot a whole lot easier! In the same way, affinity tags make it easier to work with the proteins that they are attached to.

The interacting partner molecule is conjugated to resin or agarose beads (Figure 1) that are poured to make a column, or can be purchased in prepackaged columns.

In practice, affinity purification typically consists of three steps:

·         Loading the protein onto the column.

·         Washing away contaminating proteins.

·         Eluting the protein of interest off of the column.

 Figure 2. Amino acids classified by their properties.

Ion exchange purification

How do you purify a protein that lacks an affinity tag? Alternatively, how do you further purify a protein that has already undergone affinity purification?

Ion exchange purification uses a protein’s electrostatic charge to purify it away from other contaminating biomolecules. This powerful technique takes advantage of a unique and intrinsic feature of each protein – their charge – in the purification process.

Ion exchange purification is a great way to purify a protein that lacks an affinity tag – either because it is a native protein or because you have already cut off the affinity tag.

Recall that proteins are strings of individual amino acids. A few amino acids are either positively (arginine, lysine, and histidine) or negatively (aspartate and glutamate) charged (Figure 2). The charge of the total protein is the summation of the charges on each amino acid within the protein.

Furthermore, the charge on each of these amino acids will depend on the pH of the buffer that you are using. By choosing an appropriate pH for your buffer, you can make your protein of interest either positively charged or negatively charged.

If your protein is positively charged in a given buffer, you would be performing what is known as cation exchange chromatography.

Figure 3. Negatively charged target proteins (purple) bind to positively charged agarose beads whereas positively charged contaminating proteins (pink) wash through (column 2). Target proteins are then eluted with a high salt buffer (column 3).

In this process, you would load your protein onto a negatively charged column in a buffer with low salt, wash the column, then elute your protein by increasing the salt concentration.

If your protein is negatively charged in a given buffer, then you would be doing what is called anion exchange chromatography using a positively charged column and the same steps described above for cation exchange chromatography (Figure 3).

Hydrophobic interaction purification  

Hydrophobic interaction purification uses an intrinsic property of proteins and their constituent amino acids – hydrophobicity - to purify them away from other biomolecules. Hydrophobicity means water hating and is the energetic cost of interacting with water molecules in solution.

Figure 4. Hydrophobic target proteins (purple with orange hydrophobic patch) bind to hydrophobic agarose beads (column 2) and are then eluted with a low salt buffer (column 3).

Charged amino acids are hydrophilic (“water loving”) and it is energetically favorable for them to interact with water.

However, there is an energetic penalty for hydrophobic residues to interact with water (Figure 2). You can think of these amino acids like grease or oil – they don’t like to mix with water!

Specific proteins have distinct hydrophobicities based on both their amino acid composition and on the ordering of those amino acids into the protein’s primary, secondary, and tertiary structures.

We can take advantage of a protein’s hydrophobicity to bind them to a hydrophobic column. Proteins will bind to hydrophobic columns in high salt conditions which promotes even weak hydrophobic interactions between proteins and the column surface (Figure 4).

After washing the protein, they can then be eluted by lowering the salt enough so that protein-solvent interactions become more energetically favorable compared to protein-column interactions.

Size exclusion purification

Size exclusion chromatography is a strategy that purifies proteins according to their size. In this approach proteins flow through a column of agarose beads with different mesh pore sizes. Large proteins flow through the column first followed by smaller proteins (Figure 5).

Size exclusion chromatography is unlike the purification strategies described above in that there is no discrete loading, washing, and elution steps. In contrast, the protein solution travels through a series of mesh beads with various pore sizes.

At extreme conditions, larger proteins will not fit into any bead pores, and it’s travel distance through the beads will be shorter, meaning it will elute earlier.

Unlike larger proteins, smaller ones will fit into every bead pore which will mean a longer travel distance and have a longer elution time.

A medium protein will travel through the beads with larger pore sizes, but not those with smaller pore sizes, meaning its travel distance and elution time will be between the large and small protein.

In this way, different proteins will elute according to their size (Figure 5).

As you can see, size exclusion chromatography is a very important protein purification technique!