Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues, or whole organisms. Protein purification is vital for the specification of the function, structure, and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Ideally, to study a protein of interest, it must be separated from other components of the cell so that contaminants will not interfere in the examination of the protein of interest's structure and function. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity, and biological activity. The pure result may be termed protein isolate.
Purpose The protein manufacturing cost remains high and there is a growing demand to develop cost efficient and rapid protein purification methods. Understanding the different protein purification methods and optimizing the downstream processing is critical to minimize production costs while maintaining the quality of acceptable standards of homogeneity. Protein purification is either preparative or analytical.
Preparative purifications aim to produce a relatively large quantity of purified proteins for subsequent use. Examples include the preparation of commercial products such as enzymes (e.g. lactase), nutritional proteins (e.g. soy protein isolate), and certain biopharmaceuticals (e.g. insulin). Several preparative purification steps are often deployed to remove bi-products, such as host cell proteins, which pose a potential threat to the patient's health.
Analytical purification produces a relatively small amount of a protein for a variety of research or analytical purposes, including identification, quantification, and studies of the protein's structure, post-translational modifications, and function. Each step of a protein purification scheme is monitored and takes into consideration purification levels and yield. A high purification level and a poor yield leaves hardly any protein with which to experiment. On the other hand, a high yield with low purification levels leaves many contaminants (proteins other than the one interest) which interfere with research purposes.
Preliminary steps
Extraction If the protein of interest is not secreted by the organism into the surrounding solution, the first step of each purification process is the disruption of the cells containing the protein. Depending on how fragile the protein is and how stable the cells are, one could, for instance, use one of the following methods: i) repeated freezing and thawing, ii) sonication, iii) homogenization by high pressure (French press), iv) homogenization by grinding (bead mill), and v) permeabilization by detergents (e.g. Triton X-100) and/or enzymes (e.g. lysozyme). Finally, the cell debris can be removed by differential centrifugation, which is a procedure where the homogenate is centrifuged at low speed, then again at a greater force to yield a pellet consisting of nuclei and supernatant. This yields several fractions of decreasing density where more discriminating purification techniques are applied to one fraction.
Also, proteases are released during cell lysis, which will start digesting the proteins in the solution. If the protein of interest is sensitive to proteolysis, it is recommended to proceed quickly, and to keep the extract cooled, to slow down the digestion. Alternatively, one or more protease inhibitors can be added to the lysis buffer immediately before cell disruption. Sometimes it is also necessary to add DNAse in order to reduce the viscosity of the cell lysate caused by a high DNA content.
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 inertia of each particle yields a force in the direction of the particles velocity 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.
Non-compacted particles remain mostly in the liquid called "supernatant" and can be removed from the vessel thereby separating the supernatant from the pellet. The rate of centrifugation is determined by the angular acceleration applied to the sample, typically measured in comparison to the g-force. 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 a silica-based density gradient media, like 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 center 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 further. 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. In biochemistry, ultracentrifugation is valuable for separating biomolecules and analyzing their physical properties.
Purification strategies
The choice of starting material is key to the design of a purification process. In a plant or animal, a particular protein usually is not distributed homogeneously throughout the body; different organs or tissues have higher or lower concentrations of the protein. The use of only the tissues or organs with the highest concentration decreases the volumes needed to produce a given amount of purified protein. If the protein is present in low abundance, or if it has a high value, scientists may use recombinant DNA technology to develop cells that will produce large quantities of the desired protein (this is known as an expression system). Recombinant expression allows the protein to be tagged, e.g. by a His-tag or Strep-tag to facilitate purification, reducing the number of purification steps required.
Analytical purification generally utilizes three properties to separate proteins. First, proteins may be purified according to their isoelectric points by running them through a pH-graded gel or an ion exchange column. Second, proteins can be separated according to their size or molecular weight via size exclusion chromatography or by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis. Proteins are often purified by using 2D-PAGE and are then analysed by peptide mass fingerprinting to establish the protein identity. This is very useful for scientific purposes and the detection limits for protein are nowadays very low and nanogram amounts of protein are sufficient for their analysis. Thirdly, proteins may be separated by polarity/hydrophobicity via high-performance liquid chromatography or reversed-phase chromatography.
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. Proteins are typically detected as they are coming off the column by their absorbance at 280 nm. Many different chromatographic methods exist:
Precipitation and differential solubilization
Most proteins require some salt to dissolve in water, a process called salting in. As the salt concentration is increased, proteins can precipitate, a process called salting out which involves changing protein solubility. For example, in bulk protein purification, a common first step to isolate proteins is precipitation with ammonium sulfate (NH4)2SO4. This is performed by adding increasing amounts of ammonium sulfate and collecting the different fractions of precipitated protein. Subsequently, ammonium sulfate can be removed using dialysis (separating proteins from small molecules through a semipermeable membrane). During the ammonium sulfate precipitation step, hydrophobic groups present on the proteins are exposed to the atmosphere, attracting other hydrophobic groups; the result is the formation of an aggregate of hydrophobic components. In this case, the protein precipitate will typically be visible to the naked eye. One advantage of this method is that it can be performed inexpensively, even 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 fraction 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 sulfate (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 complete purification.
Size exclusion (gel-filtration chromatography)
Chromatography can be used to separate protein in solution or denaturing conditions by using porous gels. This technique is a more discriminating separation and 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 eluent (solvent) before being collected at the other end of the column of gel. Larger molecules (or proteins) will travel through less volume and elute prior to smaller molecules.
In the context of protein purification, the eluent 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.