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Protein Purification

Written by The Protein Man | Aug 19, 2022 4:30:00 PM

Protein Purification

When investigating a particular protein of interest, the first step is to separate it from the non-protein components and from all the other proteins in the complex sample mixture (cell, tissues, or whole organisms) by exploiting differences in size, physical and chemical properties, binding affinity, and biological activities of individual proteins. 

Basically, protein purification allows researchers to identify and examine the properties of the protein of interest, including its structure, function, and interactions. When classified according to purpose, protein purification can either be preparative or analytical. 

In preparative purification, large quantities of a purified protein or isolate are produced for subsequent use while analytical purification produces a relatively small amount of a particular protein isolate for analytical or research purposes. Several enzymes, nutritional proteins, and biopharmaceutical products are produced via preparative purification, while pepsin, insulin, antibodies, and urease are usually produced through analytical purification.

 

Protein Purification: A Step-by-Step Process

Here are the steps involved in isolating a particular protein of interest.

Extraction

Unless the protein of interest is secreted by the organism into the surrounding solution, the cells and tissues containing the tissues and cells must be disrupted to release the protein of interest as a lysate. Tissue and cell are disrupted, and lysate are prepared using one of the following lysis methods:

  • Freezing and thawing
  • Homogenization by grinding
  • Sonication
  • French press
  • Permeabilization by detergents or enzymes

The most appropriate lysis method is largely dependent on the nature of the cells and tissues. Generally, the more resistant cells and tissues require more rigorous lysis protocols.

Since proteases are released during cell lysis, you may need to proceed quickly, keep the extract cooled, and add a cocktail of protease inhibitors to the lysis buffer solution prior to cell disruption and lysis to prevent proteolysis degradation of the protein of interest. 

Clarification

After extraction, you’ll want to remove all the debris and contaminants (fragmented cell membranes, organelles, insoluble proteins, etc.) from the sample solution and recover only the proteins you want to purify. Basically, a clean medium of particulates can be achieved through various methods such as precipitation, centrifugation, and filtration. 

Precipitation with ammonium sulfate is commonly used in bulk protein purification since it is relatively inexpensive, even if you’re working with large volumes. Through the addition of increasing amounts of ammonium sulfate, the hydrophobic groups in the protein aggregate to form a visible precipitate.

On the other hand, centrifugation uses centrifugal force to separate particles based on mass or density. Thus, the more massive particles with lower drag in the liquid form a pellet at the bottom of the vessel while non-compacted particles remain mostly in the supernatant after a sample is spun.

Recovery

There are several strategies that can be used to successfully isolate the protein of interest from the extract. There are a wide variety of protein extraction reagents, kits, and protein chromatography widely available from commercial sources.

  • Proteins can be isolated based on their size or molecular weight using techniques such as size exclusion chromatography, SDS-PAGE, or 2D-PAGE analysis.
  • Protein can be isolated based on their ionic characteristics by running cationic or anionic chromatography.
  • Protein can be isolated based on hydrophobicity of the protein of interest by running adsorption chromatography
  • Proteins are also purified using affinity of the target protein to certain binding molecules, such binding molecule may either be enzyme substrate or other molecules, often referred to as affinity ligands.
  • They can be separated based on their isoelectric points by running them through an ion-exchange column or pH-graded gel.
  • They can be purified according to their polarity using high-performance liquid chromatography (HPLC) or reversed-phase chromatography.

Elution

The protein of interest is extracted by washing all the non-specific binding on the column using an appropriate elution buffer. In this step, the pH of the column is altered to neutralize and extract the charged functional groups of proteins in high concentrations.

Note: The number and length of washes are usually determined by the type of column used.

Concentration

Finally, the recovered protein is concentrated via lyophilization or ultrafiltration. Lyophilization is commonly used to remove all the volatile components in the solution after an HPLC run. This method should only be used when the solution contains nothing but the protein of interest.

Alternatively, ultrafiltration uses semi-permeable membranes to concentrate a protein solution. By forcing water and small molecules to pass through the membrane, the protein and other high molecular weight solutes are left in the retentate.