Protein Purification is essential to understanding the structure and function of various proteins. It is also important for the development of clinical biosensors, which rely on stocks of pure protein as detection agents. There are many methods of protein purification, which are discussed below along with how protein purification works, the steps involved, and how to increase yield in your protein purification protocol.
How Protein Purification works
Protein purification is the process of enriching the population of a single target protein from a sample of many mixed proteins. The reason we say enriched rather than isolated is because it is difficult to obtain a perfectly pure population of a single protein from a mixed sample. However, a pure population is still the goal, and we can get quite close.
Protein purification works by taking advantage of the properties that make the target protein unique or different from other proteins in the mixed sample. These properties can be electrostatic (based on isoelectric point and charge depending on buffer pH), solubility in different salt concentrations, size, and specific binding affinity. Many of these properties can be targeted throughout a protein purification protocol, and are often used sequentially to obtain increasingly enriched populations of target.
The steps involved in protein purification
The first step in protein purification is to release the proteins from any cellular membranes that they might be sequestered inside of. Differential centrifugation can be used to obtain an enriched population of target cells or cellular organelles. These cells or cell organelles will then need to be lysed (breaking their membranes) in order to release the proteins. Cell lysis can be achieved with chemical detergents or with physical methods such as sonication. After lysis, the sample can be cleaned up with gradient centrifugation in order to remove cell debris. Keep in mind that these steps are rough cleanup steps. The fine-tooth comb of protein purification comes later.
There are a handful of commonly used protein purification methods:
- Salting out (solubility)
- As salt concentration increases, proteins with lower solubility will precipitate out of solution.
- Dialysis (size)
- Dialysis tubing or membranes can be purchased with various pore sizes based on molecular weight (Daltons or KiloDaltons). Proteins below the molecular weight cutoff (MWCO) will diffuse from the sample to the dialysate. Larger proteins above the MWCO will be retained within the dialysis membrane.
- Gel-filtration chromatography (size)
- A porous resin in prepared and the protein solution passes through it. The smaller proteins enter into the beads and take a slower path through the resin. The larger proteins move in the spaces between the beads and are eluted first.
- Ion-exchange chromatography (charge)
- This is based on the isoelectric point of the protein, which is determined by the amino acid composition. If one protein has an overall positive charge at pH 7, and another protein has an overall negative charge at pH 7, then the positively charged protein will be attracted to a negatively charged column and can be separated from the other protein.
- Affinity Chromatography (binding affinity)
- This takes advantage of binding pockets in the protein and recognition of certain molecules. Some examples include biotin-Streptavidin affinity, His tags, protein A/G chromatography, and antibody-antigen interactions.
After completing a protein purification method it is a good idea to verify the identity of the enriched protein. Some verification techniques include gel electrophoresis and mass spectrometry. Unfortunately, both of these techniques rely on a knowledge of the exact mass of the target protein. Additionally, activity assays may be performed with proteins that have known activity with certain molecules or other proteins.
Increasing protein purification yield
High Pressure Liquid Chromatography is another chromatography technique that could improve your protein purification yield because it enables finer resolution between sample fractions. The columns used have more reaction sites, and are able to separate a mixed solution into clearly divided fractions. This means less overlap between proteins, decreased loss, and cleaner fractions.
Combining protein purification methods to create a protocol that moves from purifying based on general properties like size followed by charge, and ending with affinity chromatography can result in an increasingly enriched sample of target protein. This stepwise approach can improve yield by slowly concentrating the protein of interest and increasing the chances of removing unwanted proteins.
It is always important to approach protein purification with a good understanding of the properties of your protein of interest. A good understanding of your protein’s isoelectric point and behavior in varying salt concentration and pH will prove to be very useful during the purification process.
Once a protein is enriched and verified it can be further studied to gain more insight into structure and function. From there it might be used to create a bioassay to be used in clinical diagnosis of disease. All discovery and innovation begins with purification.