The Importance of Protein Purification
Before a protein or other biological macromolecule can be rigorously studied from a structural and functional basis, it must be purified. The problems that can arise during protein purification become clear when one considers that a single protein has to be purified from a mixture of as many 10,000 other cellular or tissue proteins, each of which is made up of the same constituent amino acids. Proteins differ in size (how many amino acids), charge (how many positively and negatively charged amino acids), sequence, and presence of specific binding sites on the proteins. Any technique that could be used to purify protein must be based on these inherent differences.
Once the protein is purified, it must be analyzed, typically by a spectral or electrophoretic technique. Protein purification is a series of processes intended to isolate and purify a single protein or complex from cells, tissues, or whole organisms. Protein purification is vital for the characterization of the function, structure, and interactions of the protein of interest. Separation steps usually exploit differences in protein size, physical-chemical properties, binding affinity, and biological activity.
Preparative vs. Analytical Protein Purification
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). Many steps and much quality control is required to remove other host proteins and other biomolecules, 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, structural characterization, and studies of the protein’s structure, post-translational modifications, and function.
Choosing the Starting Material
The choice of a starting material is key to the design of a purification process. In plants or animals, a particular protein usually isn’t distributed homogeneously throughout the organism; different organs or tissues have higher or lower concentrations of the protein. The use of 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. These techniques will be discussed in greater detail in Chapter 5.
Cell Disruption and Protein 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, one of several techniques could be used including repeated freezing and thawing, sonication, homogenization by high pressure (French press), homogenization by grinding (bead mill), and permeabilization by detergents (e.g. Triton X-100) and/or enzymes (e.g. lysozyme).
Finally, the cell debris can be removed by centrifugation so that the proteins and other soluble compounds remain in the supernatant. Also proteases are released during cell lysis, which will start digesting the proteins in the solution. As the protein of interest may be sensitive to proteolysis, it is important to proceed quickly and conduct many steps at low temperatures to reduce unwanted proteolysis. 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.
Centrifugation and Density Gradient Separation
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 particle’s 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.
In 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 the 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 toward the bottom of the tube faster than lighter material. After separating the protein/particles, the gradient is then fractionated and collected.
Salt Precipitation and Dialysis
In bulk protein purification, a common first step to isolate proteins is precipitation using a salt such as ammonium sulfate (NH4)2SO4. Ammonium sulfate is often used as it is highly soluble in water, has relative freedom from temperature effects, and typically is not harmful to most proteins. Proteins are precipitated by (NH4)2SO4 in their native state, which is important if you need the protein for structure/function studies.
Furthermore, ammonium sulfate can be removed by dialysis as described in Figure 3.2. The process of dialysis separates dissolved molecules by their size. The biological sample is placed inside a closed membrane, where the protein of interest is too large to pass through the pores of the membrane, but through which smaller ions can easily pass. As the solution comes to equilibrium, the ions become evenly distributed throughout the entire solution, while the protein remains concentrated in the membrane. This reduces the overall salt concentration of the suspension.
Chromatographic Separation Techniques
Chromatography is used in almost all protein purification methods and is the key that allows the separation of a given protein from the 1000s of different proteins in cells and tissues. The separation of proteins on a chromatography column depends on the type of column and chemical/physical properties of the molecule. There are four main types used of chromatographies used to separate proteins:
Size Exclusion Chromatography
Size exclusion chromatography, also known as gel filtration chromatography, is a low-resolution isolation method that involves the use of beads that have tiny “tunnels” in them that each have a precise size. The size is referred to as an “exclusion limit,” which means that molecules above a certain molecular weight will not fit into the tunnels. Molecules with sizes larger than the exclusion limit do not enter the tunnels and pass through the column relatively quickly by making their way between the beads. Smaller molecules, which can enter the tunnels, do so, and thus, have a longer path that they take in passing through the column. Because of this, molecules larger than the exclusion limit will leave the column earlier, while smaller molecules that pass through the beads will elute from the column later.
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 compounds (anions), while cation exchange resins have a negative charge and are used to separate positively charged molecules (cations). 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.
Affinity Chromatography
Affinity Chromatography is a separation technique based upon molecular conformation, which frequently utilizes application specific resins. These resins have ligands (small molecules) attached to their surfaces which are specific for and will bind with 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 unretained.
Hydrophobic Interaction Chromatography
HIC media is similar to reverse phase chromatography in which a matrix like silica (very polar with exposed OH groups) is derivatized with ester or ether links from the silica surface hydroxyl OHs to nonpolar molecules, usually containing 8 or 18 carbons in the acyl or alkyl chain. Proteins with exposed hydrophobic groups would preferentially bind to the bead. The interactions of the protein with the derivatized beads are increased by adding high concentrations of salt to the aqueous solution, making water effectively more polar. This would shift the equilibrium towards binding of the surface-exposed nonpolar region on the protein to the nonpolar C8 or C18 chains.
Denaturing Protein Purification Techniques
For proteins that are not soluble in water, such as transmembrane proteins that span cell membranes and large fibrous proteins, detergents like sodium dodecyl sulfate (SDS) can be used to unfold the proteins and keep them in solution during purification. Milder detergents like Triton X-100 can also be used to retain the protein’s native conformation.
High Performance Liquid Chromatography (HPLC) and Fast Protein Liquid Chromatography (FPLC)
HPLC is a form of chromatography applying high pressure to drive the solutes through the column faster than using gravity-forced flow of solvent through the column. The most common form is “reversed phase” HPLC, where the column packing material is hydrophobic. The proteins are eluted by a gradient of water and increasing amounts of an organic solvent, such as acetonitrile. The proteins elute according to their hydrophobicity.
FPLC is a form of liquid chromatography that is often used to analyze or purify mixtures of proteins. In FPLC, the mobile phase is an aqueous solution, or “buffer”. The buffer flow rate is controlled by a positive-displacement pump and is normally kept constant, while the composition of the buffer can be varied by drawing fluids in different proportions from two or more external reservoirs. The stationary phase is a resin composed of beads, usually of cross-linked agarose, packed into a cylindrical glass or plastic column.
Quantitative Analysis During Protein Purification
During the protein purification process, it is necessary to have a quantitative system to determine the total amount and concentration of total and target protein at each step, the biological activity of the target protein, and its overall purity. This will help guide and optimize the purification method being developed.
Key parameters that are quantitatively evaluated include:
- Total Protein: The total amount of protein in the sample, calculated by multiplying the concentration by the total volume.
- Total Activity: The total enzymatic or biological activity of the target protein, calculated by multiplying the activity per unit volume by the total volume.
- Specific Activity: The ratio of total activity to total protein, providing a measure of the purity and specific activity of the target protein.
- Yield: The percentage of target protein activity retained at each purification step compared to the initial sample.
- Purification Level: The fold-increase in specific activity compared to the initial sample, providing a measure of the overall purity achieved.
Monitoring these parameters at each step allows ineffective separation techniques to be disregarded and more effective purification methods to be adopted to maximize yield and retain biological activity.
Electrophoretic Separation Techniques
In addition to chromatography, electrophoretic techniques are also commonly used for protein purification and analysis. These methods leverage differences in size, shape, and charge of proteins to separate them in an electric field.
Polyacrylamide Gel Electrophoresis (PAGE)
Polyacrylamide gel electrophoresis (PAGE) is one of the most widely used techniques to characterize complex protein mixtures. Proteins are separated based on their size and charge, either in their native state (native PAGE) or under denaturing conditions using SDS-PAGE, where the proteins are coated with the anionic detergent SDS to give them a uniform negative charge.
Isoelectric Focusing (IEF)
Isoelectric focusing is a technique that separates proteins based on their isoelectric point (pI) – the pH at which the protein has no net charge. Proteins are loaded onto a gel with an established pH gradient, and under an applied electric field, they will migrate to the region of the gel where the pH matches their pI, where they will then stop migrating.
Two-Dimensional Gel Electrophoresis (2D-PAGE)
Two-dimensional gel electrophoresis combines isoelectric focusing in the first dimension to separate proteins by pI, followed by SDS-PAGE in the second dimension to separate by molecular weight. This powerful technique can resolve thousands of proteins in a single experiment and is often coupled with mass spectrometry for protein identification.
Western Blotting
After separating proteins by electrophoresis, the Western blot technique is commonly used to detect and analyze specific proteins of interest. Proteins are transferred from the gel to a membrane, where they can be probed with specific antibodies and detected using various reporter systems like enzymes or fluorescent labels.
Peptide Synthesis and Sequencing Techniques
For proteins that are difficult to purify or express in sufficient quantities, chemical peptide synthesis can be a useful approach. Solid-phase peptide synthesis allows rapid assembly of peptides by sequential coupling of amino acid derivatives on an insoluble resin support. This technique enables the incorporation of unnatural amino acids and modification of the peptide backbone.
Protein sequencing techniques like Edman degradation can also provide valuable information about the N-terminal amino acid sequence of a purified protein, which can aid in its identification.
Mass Spectrometry Applications
Mass spectrometry has become an indispensable tool for protein characterization and identification. Techniques like MALDI-TOF MS and LC-MS/MS enable accurate mass determination, identification of post-translational modifications, and de novo sequencing of peptides derived from proteolytic digestion of purified proteins.
Structural Characterization Techniques
In addition to biochemical and functional studies, purified proteins can also be subjected to structural analysis techniques like X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. These methods provide detailed 3D structures that are crucial for understanding a protein’s mechanism of action and potential for targeted drug development.
Overall, the field of protein purification is crucial for unlocking the secrets of the proteome and enabling in-depth studies of protein structure, function, and interactions. By leveraging the diverse array of separation, detection, and characterization techniques, researchers can obtain highly pure, well-characterized proteins that serve as the foundation for groundbreaking discoveries in biology and medicine.
