This article throws light upon the four methods of protein purification.
The four methods of protein purification are: (1) Extraction (2) Precipitation and Differential Solubilisation (3) Ultracentrifugation and (4)Chromatographic Methods.
The methods used in protein purification, can roughly be divided into analytical and preparative methods.
The distinction is not exact, but the deciding factor is the amount of protein, that can practically be purified with that method. Analytical methods aim to detect and identify a protein in a mixture, whereas preparative methods aim to produce large quantities of the protein for other purposes, such as structural biology or industrial use. In general, the preparative methods can be used in analytical applications, but not the other way around.
Method # 1. Extraction:
Depending on the source, the protein has to be brought into solution by breaking the tissue or cells containing it. There are several methods to achieve this; Repeated freezing and thawing, sonication, homogenization by high pressure or permeabilization by organic solvents. The method of choice depends on how fragile the protein is and how sturdy the cells are.
After this extraction process soluble protein will be in the solvent, and can be separated from cell membranes, DNA, etc. by centrifugation. The extraction process also extracts proteases, which will start digesting the proteins in the solution. If the protein is sensitive to proteolysis, it is usually desirable to proceed quickly, and keep the extract cooled, to slow down proteolysis.
Method # 2. Precipitation and Differential Solubilisation:
In bulk protein purification, a common first step to isolate proteins is precipitation with ammonium sulphate (NH4)2SO4. This is performed by adding increasing amounts of ammonium sulphate and collecting the different fractions of precipitate protein. One advantage of this method is that it can be performed inexpensively with very large volumes.
The first proteins to be purified are water-soluble proteins. Purification of integral membrane proteins requires disruption of the cell membrane in order to isolate any one particular protein from others that are in the same membrane compartment. Sometimes a particular membrane traction can be isolated first, such as isolating mitochondria from cells before purifying a protein located in a mitochondrial membrane.
A detergent such as sodium dodecyl sulphate (SDS) can be used to dissolve cell membranes and keep membrane proteins in solution during purification; however, because SDS causes denaturation, milder detergents such as Triton X-100 or CHAPS can be used to retain the protein’s native conformation during purification.
Method # 3. Ultracentrifugation:
Centrifugation is a process that uses centrifugal force to separate mixtures of particles of varying masses or densities suspended in a liquid. When a vessel (typically a tube or bottle) containing a mixture of proteins or other particulate matter, such as bacterial cells, is rotated at high speeds, the angular momentum yields an outward force to each particle that is proportional to its mass.
The tendency of a given particle to move through the liquid because of this force is offset by the resistance the liquid exerts on the particle. The net effect of “spinning” the sample in a centrifuge is that massive, small, and dense particles move outward faster than less massive particles or particles with more “drag” in the liquid.
When suspensions of particles are “spun” in a centrifuge, a “pellet” may form at the bottom of the vessel that is enriched for the most massive particles with low drag in the liquid. The remaining, non-compacted particles still remaining mostly in the liquid are called the “supernatant” and can be removed from the vessel to separate the supernatant from the pellet.
The rate of centrifugation is specified by the angular acceleration applied to the sample, typically measured in comparison to the g. If samples are centrifuged long enough, the particles in the vessel will reach equilibrium wherein the particles accumulate specifically at a point in the vessel where their buoyant density is balanced with centrifugal force. Such an “equilibrium” centrifugation can allow extensive purification of a given particle.
Sucrose gradient centrifugation:
A linear concentration gradient of sugar (typically sucrose glycerol, or Percoll) is generated in a tube such that the highest concentration is on the bottom and lowest on top. A protein sample is then layered on top of the gradient and spun at high speeds in an ultracentrifuge. This causes heavy macromolecules to migrate towards the bottom of the tube faster than lighter material.
During centrifugation in the absence of sucrose, as particles move farther and farther from the centre of rotation, they experience more and more centrifugal force (the further they move, the faster they move). The problem with this is that the useful separation range within the vessel is restricted to a small observable window.
Spinning a sample twice as long does not mean the particle of interest will go twice as far; in fact, it will go significantly farther. However when the proteins are moving through a sucrose gradient, they encounter liquid of increasing density and viscosity.
A properly designed sucrose gradient will counteract the increasing centrifugal force, so the particles move in close proportion to the time they have been in the centrifugal field. Samples separated by these gradients are referred to as “rate zonal” centrifugations. After separating the protein/particles, the gradient is then fractionated and collected.
Method # 4. Chromatographic Methods:
Usually a protein purification protocol contains one or more chromatographic steps. The basic procedure in chromatography is to flow the solution containing the protein through a column packed with various materials. Different proteins interact differently with the column material, and can thus be separated by the time required to pass the column, or the conditions required to elute the protein from the column. Usually proteins are detected as they are coming off the column by their absorbance at 280 nm.
Many different chromatographic methods exist:
1. Size Exclusion Chromatography:
Chromatography can be used to separate protein in solution or denaturing conditions by using porous gels. This technique is known as size exclusion chromatography. The principle is that smaller molecules have to traverse a larger volume in a porous matrix. Consequentially, proteins of a certain range in size will require a variable volume of eluant (solvent) before being collected at the other end of the column of gel.
In the context of protein purification, the eluant is usually pooled in different test tubes. All test tubes containing no measurable trace of the protein to purify are discarded. The remaining solution is thus made of the protein to purify and any other similarly-sized proteins.
2. Ion Exchange Chromatography:
Ion exchange chromatography separates compounds according to the nature and degree of their ionic charge. The column to be used is selected according to its type and strength of charge. Anion exchange resins have a positive charge and are used to retain and separate negatively charged com pounds, while cation exchange resins have a negative charge and are used to separate positively charged molecules.
Before the separation begins a buffer is pumped through the column to equilibrate the opposing charged ions. Upon injection of the sample, solute molecules will exchange with the buffer ions as each competes for the binding sites on the resin. The length of retention for each solute depends upon the strength of its charge.
The most weakly charged compounds will elute first, followed by those with successively stronger charges. Because of the nature of the separating mechanism, pH, buffer type, buffer concentration, and temperature all play important roles in controlling the separation. Ion exchange chromatography is a very powerful tool for use in protein purification and is frequently used in both analytical and preparative separations.
3. Affinity Chromatography:
Affinity Chromatography is a separation technique based upon molecular conformation, which frequently utilizes application specific resins. These resins have ligands attached to their surfaces which are specific for the compounds to be separated. Most frequently, these ligands function in a fashion similar to that of antibody-antigen interactions. This “lock and key” fit between the ligand and its target compound makes it highly specific, frequently generating a single peak, while all else in the sample is un-retained.
Many membrane proteins are glycoproteins and can be purified by lectin affinity chromatography. Detergent solubilized proteins can be allowed to bind to a chromatography resin that has been modified to have a covalently attached lectin.
Proteins that do not bind to the lectin are washed away and then specifically bound glycoproteins can be eluted by adding a high concentration of a sugar that competes with the bound glycoproteins at the lectin binding site. Some lectins have high affinity binding to oligosaccharides of glycoproteins that is hard to compete with sugars, and bound glycoproteins need to be released by denaturing the lectin.
4. Metal Binding:
A common technique involves engineering a sequence of 6 to 8 histidines into the C-terminal of the protein. The polyhistidine binds strongly to divalent metal ions such as nickel and cobalt. The protein can be passed through a column containing immobilized nickel ions, which binds the polyhistidine tag. All untagged proteins pass through the column.
The protein can be eluted with imidazole, which competes with the polyhistidine tag for binding to the column, or by a decrease in pH (typically to 4.5), which decreases the affinity of the tag for the resin. While this procedure is generally used for the purification of recombinant proteins with an engineered affinity tag (such as a 6xHis-tag or Clontech’s HAT tag), it can also be used for natural proteins with an inherent affinity tor divalent cations.
5. Immunoaffinity Chromatography:
Immunoaffinity chromatography uses the specific binding of an antibody to the target protein to selectively purify the protein. The procedure involves immobilizing an antibody to a column material, which then selectively binds the protein, while everything else flows through. The protein can Ix eluted by changing the pH or the salinity. Because this method does not involve engineering in a tag, it can be used for proteins from natural sources.
High performance liquid chromatography or high pressure liquid chromatography is a form of chromatography applying high pressure to drive the solutes through the column faster. This means that the diffusion is limited and the resolution is improved. The most common form is “reversed phase” HPLC, where the column material is hydrophobic.
The proteins are eluted by a gradient of increasing amounts of an organic solvent, such as acetonitrile. The proteins elute according to their hydrophobicity. After purification by HPLC the protein is in a solution that only contains volatile compounds, and can easily be lyophilized. HPLC purification frequently results in denaturation of the purified proteins and is thus not applicable to proteins that do not spontaneously refold.