Advancements in recombinant protein expression technologies allow the production of pretty much any protein or specific protein domains, including modifications or mutations to the native amino acid sequence. Added to that, the ability to add a range of tag sequences to the expressed protein allows the scientist to purify and characterise the protein in a variety of ways. In some cases a protein can be purified and submitted for varied biochemical or structural studies, with little more than a two-step purification route, yielding high quality protein.
There are still a few protein scientists to be found who talk of when ion-exchange (IEX) chromatography was a key tool in a protein scientist’s armoury, with which to isolate and enrich their protein of interest, teasing apart proteins according to their surface charge, and achieving multi-peaked elution profiles to rival the Himalayas! With the wide use of affinity purification tags, the technique of IEX chromatography seems to have lost its edge. In our experience though, IEX is a relatively simple chromatography technique; useful at all stages of purification, from initial capture, through to final polishing, and provides high capacity and resolving power, under mild conditions, whilst being very economical to perform.
Chromatographic separation on the basis of charge is a very favourable tool for purifying proteins from one another, largely because each protein has a unique amino acid sequence with a different number and type of ionizable amino acid side chains. Protein molecules are amphoteric, containing both acidic and basic groups, existing as zwitterions over a certain pH range. The pH at which a protein has no net charge is called its Isoelectric point (pI). At a pH below the pI, a protein will have a net positive charge (so being able to bind to a cation exchange resin), and at a pH above the pI, a protein will have a net negative charge (so being able to bind to an anion exchange resin). As determined by their pI, proteins can thus be encouraged to bind to both anion and cation exchange resins over a certain pH range. Binding takes place typically under low ionic strength, and the proteins are then eluted differentially, usually by applying a salt gradient (increasing ionic strength), or by a pH gradient (altering the charge state of the protein).
Determining the pI of your protein of interest, from which to work out an IEX strategy, is as easy as ‘pie’! The wonderful Henderson-Hasselbach equation is used to compute protein charge at different pH values (based on its amino acid sequence), until one is found where it has a net zero charge. Online tools take the hard work out of the mathematics. The commonly used ProtParam (hosted by ExPASy), gives a host of useful physicochemical properties from the submitted amino acid sequence.
Having a theoretical pI to hand is one thing, but confirming it experimentally is often recommended. Likely more than a few protein scientists have chosen a pH, buffer, and either cation or anion exchange column based solely on the theoretical pI, and then ended up scratching their heads wondering why their protein doesn’t behave as expected. However, pI calculations based on amino acid sequence do not consider the 3D structure of the protein and whether various surface charges on the protein are inaccessible or unevenly arranged over the molecule. Post-translational modifications too can impact the net charge on a protein, e.g. phosphorylation, or addition of charged monosaccharides, and even non-charged glycans can potentially shield surface charged groups from the exterior environment.
pH scouting experiments are therefore invaluable in determining optimal pH, buffer conditions, and ion exchange resins to use for purification of a protein of interest, as well as identifying conditions for purification from other protein contaminants. In our experience, however eager we may be to move a project forward, some time spent on experimental feasibility work early on can often save headaches further down the line. There is no substitution to getting to know your protein!
As an example, we worked with a protein that, based on its sequence, would be expected to be positively charged at neutral pH, and expected to bind to a cation exchange resin. During expression however, we were selecting for a heavily phosphorylated species, giving it a net negative charge, which then purified beautifully on an anion exchange resin. In another example, and to show the resolving power of IEX, we were able to separate a mono-phosphorylated protein from its di- and tri-phosphorylated siblings. Using a strong anion exchange resin, of fine particle size, a shallow salt gradient was able to tease these three species apart. This was important as ultimately, the mono-phosphorylated protein was the only species that crystallised and yielded a structure. Not a bad result for the humble ion exchanger! Thus ion-exchange is a powerful technique and still has much to offer as a tool for protein purification. So, where protein purification is concerned it is well worth keeping all ‘ions’ in the fire!
To find out more about how Peak Proteins could help you with your protein purification workflows, have a look at our protein expression and purification service page or contact us directly at firstname.lastname@example.org