Proteins are the key functional component of the cells, handling most biological functions, from signaling to homeostasis, immunity, structural integrity, gene expression and to enzyme-driven chemical catalysis.
Proteins purification was at the center of the early days of the pharmaceutical and chemical industry, with processes extracting with difficulties a few grams of pure products from raw biological material. The chemical and biotech industry and modern medicine are highly reliant on a steady supply of highly purified proteins, including antibodies and enzymes.
Modern techniques are a lot more material and cost-efficient, while also allowing for higher quality protein purification. Many such procedures are also partially or fully automated, reducing the risk of error and labor costs.
There are multiple protein purification methods, of which one of the most popular and ancient is protein purification chromatography. Chromatography was one of the earliest protein purification methods, with more than a century of history by now.
Chromatographic separation can be categorized into 2 categories:
- Preparative chromatography, to separate a purified fraction of the initial mixed sample. The emphasis is on selectivity and purity.
- Analytical chromatography, to identify the nature of unknown compounds or the quantity of highly purified compounds. The emphasis is on protein analysis, characterization, and measuring accuracy.
Most protein purification chromatographies done in industrial protein production tend to be preparative, with a focus on purity and cost efficiency.
Most protein purification done in diagnostics, testing, and food production tend to be analytical, with a focus on sensibility and lowering the risk of false positives or false negatives.
When dealing with industrial production of individual proteins, protein purification methods often rely on recombinant proteins. Such types of proteins are generally produced in bioreactors and their amino acid sequences have been modified through genetic engineering.
Once modified, the desired protein expression includes either a “tag” or is fused with another protein. The expression of such tags/markers allows for easier extraction of individual proteins in a complex mix, creating a tightly controlled system for protein expression and purification.
One popular form of protein purification through chromatography is affinity chromatography. It relies on the specific interaction between a chosen ligand and a (ideally unique) protein three-dimensional configuration. For example, the interaction between an antigen and a bispecific antibody, a receptor and its activator, or an enzyme and its substrate. Because of the high specificity of the interaction, it is possible to generate high-purity protein products with this method. You can read more about this technique on our affinity chromatography page.
Another option is ion-exchange chromatography, which is a procedure that allows the separation of proteins depending on their pH.
Typically, in the first step, the protein binds to the chromatography columns. Then in successive steps, the proteins are progressively released with a change in the buffer’s pH, allowing for a splitting of different protein fractions depending on their acidity.
While this method often does not allow for the selection of a unique protein in the proteins purification process, it can greatly reduce the complexity of a sample/batch. You can read more about this technique on our ion-exchange chromatography page.
Size exclusion chromatography, also referred to as gel filtration, is a “mild” form of chromatography. Its principle is that the larger a protein (or any other molecules), the more it will be hindered in its movement inside a porous matrix of beads.
Given long enough time, this will separate proteins according to their molecular weight, as well as their actual physical size (which can vary from molecular weight depending on protein folding).
You can read more about this technique on our size exclusion chromatography page.
Another way to prepare a sample for extraction, ahead of chromatography-driven proteins purification, is protein precipitation. The method relies on different proteins having different degrees of hydrophobicity. When increasing progressively the concentration of salt/precipitation agent, interactions between proteins cause fractional precipitation. This allows the removal of a significant portion of the protein in the sample that has a different hydrophobic profile than the targeted protein.
When performing downstream protein purification, it is often necessary to exclude small molecular weight substances. This can include labeling or reducing agents or preservatives, all of which might interfere with later procedures.
A common method used is dialysis. Dialysis in enzyme purification is especially important, with salt and other impurities likely to interfere with enzymatic activity. The principle of this method is based on a semi-permeable membrane, with pores large enough to let small molecules go through but block larger molecules like proteins & enzymes.
The buffer volume on the other side of the membrane has to be much larger than the sample, creating an osmotic pressure for the small molecules to spread on the other side of the membrane, effectively removing the large majority of them from the protein sample.