Most people know that proteins are vital biological compounds and play instrumental roles in research, industrial, and medical settings. But before the identification of any specific protein that could lead to the analysis and subsequent understanding of its properties, a few things must first take place.
For one, it must be extracted from an animal or plant or separated from a complex biochemical mixture. Then it needs to be purified. These purification and extraction processes form a crucial component of many protein-specific scientific endeavors. However, the technique employed in these processes will vary based on the nature of the solution and the protein in question.
What is Protein Purification?
Protein purification, in a nutshell, is a series of procedures carried out to isolate a specific protein or proteins from either a cell or tissue mixture. A particular protein is sometimes placed in a matrix of several other protein molecules or found among non-protein biological molecules.
Whatever might be the case, researchers may only need a small protein sample for analytical reasons. On the other hand, vast amounts of purified protein may be required for commercial or industrial purposes, mainly carried out in a protein purification service.
In either case, most purification processes involve four main techniques, namely:
- Precipitation and differential solubilization
- Chromatographic methods
These four methods can be categorized into two groups, namely, preliminary steps and purification strategies. Continue reading to learn the details concerning these two categories.
Depending on the origins of the protein, it first needs to be contained in a solution by disintegrating the cells or tissue that contain the protein of interest. For this purpose, different methods come into play depending on the fragility of the protein and the stability of the containing cells. The methods used are:
- Permeabilization by detergents
- Homogenization by grinding
- Freezing and thawing repeatedly
- Homogenization by high pressure
Using these methods will leave behind cell debris which is harvested by centrifugation. Through centrifugation, the specific proteins and several other compounds are leftover in the supernatant. Then other proteins are digested using protease during the cell lysis.
Precipitation And Differential Solubilization
Precipitation is a standard process used in bulk protein purification to extraction using ammonium sulfate (NH4)2SO4. This process involves the subsequent and repeated addition of increasing amounts of ammonium sulfate and retrieving the different sections of precipitate protein. Later, the dialysis is used to eliminate the previously added ammonium sulfate. This approach comes with a key advantage- its inexpensive nature, even on a large scale.
The first purified proteins are usually water-soluble ones. However, cell membrane disruption is required before integral membrane proteins become purified, involving the use of sodium dodecyl sulfate (SDS), CHAPS, or Triton X-100.
During the high-speed rotation of a mixture comprising different proteins and particulate matter like bacterial cells, the individual inertia of each component generates a force in the particle’s velocity’s direction that’s proportional to the particle’s mass. Consequently, using a centrifuge makes small, dense, and massive particles move outward quicker than particles with more drag within the mixture. The liquid mixture usually has non-compacted particles called “supernatant” and pellets that form at the bottom. This makes the removal of the supernatant from the vessel possible. But thanks to “equilibrium” centrifugation, a given particle in the mixture can reach extensive purification.
The typical protein purification protocol consists of more than one chromatographic step. The different chromatographic methods are as follows.
Size Exclusion Chromatography
This chromatography method involves using a porous gel that helps to separate protein in solution or denaturing situations. It works based on the principle that smaller molecules will go through a smaller volume when located in a porous matrix. As a result, proteins in a particular size range will need a variable volume of solvent or eluant before collecting them at the opposite end of the gel column. For the purification of the protein, the eluant is normally collected into separate test tubes. Any protein that contains no quantifiable trace of the specific protein is discarded.
Affinity chromatography is a method of separation that depends mainly on the molecular conformation that regularly uses resins specific to the case application. The resins, in this case, have ligands bound to their surfaces that are compound-specific according to the elements intended for separation. More often than not, these ligands function in ways similar to the antibody-antigen interactions, which can be likened to a “lock and key” fit. The nature of the interaction between the ligand and the target elements renders their interaction extremely specific, frequently producing one peak. In contrast, everything else contained in the sample is un-retained.
Ion Exchange Chromatography
This method of chromatography helps separate compounds based on the degree and nature of their ionic charges. The selected column for use is chosen according to strength and type of charge. Anion exchange resins possess a positive charge and help retain and extract compounds with negative charges. On the other hand, cation exchange resins are negatively charged and employed to separate molecules with positive charges. The opposing charge ions are equalized by pumping a buffer into the column to facilitate the separation process. After the sample gets injected, sample molecules will exchange with buffer ions because they contend for the resin’s binding sites.
On the whole, protein purification methods can be analytical or preparative. Analytical purification usually aims at producing a relatively smaller quantity of protein for research reasons. On the other hand, preparative purification produces a relatively larger amount of purified proteins for use in commercial or industrial settings. Whatever approach is taken depends ultimately on the intended use of the purified protein.