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Posted on Thu, Apr 28, 2022

Protein purification: the step-by-step process

Protein purification is the processes of isolating a protein of interest from its environment. In other words, from the other natural molecules surrounding the proteins in the natural niche in a host organism, or from a cell culture grown in a laboratory. Our protein purification handbook  explains that there are several available techniques and many options to consider, but the general procedure is the same. 

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Types of Protein purification techniques

Chromatography is a widely used purification technique that utilizes columns filled with molecules chosen to separate your protein of interest. These molecules may be:

  • Nickel bound column for binding a HIS-TAG on your protein
  • Ion-exchange or cation-exchange chromatography for binding your protein based on charge 
  • Size-exclusion columns contain a range of cross-linked polymers to separate based on size
  • Affinity columns will be bound with a ligand of your protein to capture it

Labs that perform many purifications will prefer to use a high performance liquid chromatography machine (HPLC). The HPLC is costly and the procedure can be complicated, but yields good precise results that are typically reproducible. Chromatography purification can also be time consuming because it requires making reagents for each column used and many wash steps. 

This post is an excerpt from our protein purification handbook, which explains the basics of protein purification steps. Download The Basic Guide to Recombinant Protein Purification here:

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Starting the protein purification steps: sample preparation

The process starts with the preparation of the sample, which consists of cell harvesting, cell disruption (in the case our target protein is intracellular), and clarification. Cell harvesting involves separating the cells from the culture medium, usually by centrifugation or filtration. As for the cell disruption, it can be achieved through different methodologies that will be chosen depending on the host. Not all cells demonstrate the same resistance to lysis, and this should be taken into account because more rigorous protocols are needed for the more resistant cells (ex: bacterial cells, due to their cell walls).  The most widely used techniques are chemical methods (such as enzymatic methods or detergents) and physical methods such as sonication.

All methods have advantages and disadvantages, and they can never produce a 100% pure sample. This is why many protein purification techniques are said to produce an “enriched population” of a target protein rather than a pure population. Due to this, we tend to use combinations of methods, such as, for example, sonication followed by a detergent treatment. Obviously, in the case of preparing a sample of secreted proteins, cell disruption will not be needed and one step will be eliminated.

The last preparation step is the clarification, which is needed because the protein to be purified will be mixed with other elements such as membrane remains, organelles, cellular debris or insoluble proteins. This is achieved by filtration or centrifugation, which uses centrifugal force to separate the liquid fraction with dissolved soluble molecules from the heavier intact cells. The goal is to obtain a clean medium of particulates, which will allow us to carry out the separation itself. It is during these steps that we try to decrease the working volume in order to deal with more manageable volumes. However, as is often the case in industrial scenarios, the volume needs to be scaled up in order to increase production. The scaling-up process can be a tricky one as large volumes of proteins and cells can behave differently than small volumes. This is particularly true when biomagnetic separation is used in the next step, the capture step. 

 

protein purification

Isolating the protein in the capture step

The next purification step is recovery or capture, which has as a goal to isolate, concentrate and stabilize the protein of interest. Always preserving its functionality, the protein is concentrated and the most important contaminants are eliminated. This step functions as a simple and fast initial purification that will be refined later, although a high level of purity can be obtained with very specific methods.

As discussed before, affinity chromatography is, currently, the most common method for purifying recombinant proteins. We should also consider magnetic bead separation, which provides many advantages in regards to chromatography and assumes a general simplification of the process. In case of using less specific methods, an intermediate purification may occur in order to separate the protein of interest from the contaminants that linger after the clarification.

Polishing step: refining our results

The last step is polishing, although it overlaps a little with the purification and it’s not always needed. This step consists of eliminating as much of the impurities as possible, performing more chromatographies, or eliminating some elements used in the chromatography that are not needed (or even undesired) in the final product. We can do a dialysis or change the buffer in order to have the already purified final protein in adequate conditions, such as pH or salinity. Polishing is done or not depending on the desired purity grade and on the acceptable efforts to be put into the process. As already discussed, this will depend on the use of the purified protein.

Uses and importance of protein purification

Protein purification protocols have a wide range of sizes. In industry, scientific companies need to make large batches of protein. This is because companies have optimized their purification procedure by the time they are manufacturing, and they just need to produce as much quality product as possible.  In laboratories, proteins are purified on a smaller scale. Laboratories are interested in studying proteins.

There are many reasons to study proteins. Some laboratories are interested in the binding partners of proteins, they might use a technique called “tandem affinity purification” which has multiple steps. It is also important to study the kinetics of proteins, such as enzymes. Biophysicists are interested in rates associated with the structure and function of enzymes. For example, scientists are interested in learning about the kinetics of polymerases, to better understand their speed and propensity for error. Scientists are also interested in understanding the structure of proteins by x-ray crystallography. To do this technique, proteins need to be as pure as possible. Magnetic beads offer high yield through an efficient protocol.

Companies and laboratories can benefit from the simplicity of the magnetic beads technique now that magnetic separation systems come in various sizes.

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Did you find this post interesting? You can check this related post in order to learn more about protein purification steps:

 

 

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