The isolation and purification of proteins is a daunting task incapable of being distilled down to a simple set of instruction. The process is nearly always customized for the protein of interest. However, prior to initiating the purification process it is necessary to first develop an assay for the protein. For enzymes, the assay may be a simple colorimetric test. Alternatively it may involve an indirect measurement, such as an immunoassay. For the purification, the source of the protein must be identified and collecting the "raw" material must be optimized. The protein must be liberated from the source by using a homogenizer, detergent, or enzymatic process in conditions that prevent its degradation. Subsequently the protein must be captured and stabilized using one of many protein purification methodologies. Those same techniques can be used in tandem to eliminate contaminating molecules and increase the purity of the desired protein.
Stabilization of the protein during homogenization through the various stages of purification depends on the multi-component buffer system used to solubilize the protein while maintaining biological activity. Maintenance of biological activity generally requires stabilizing the protein's tertiary structure. Since protein purification by definition is in vitro, the buffer system developed by the researcher should mimic as closely as possible the in vivo conditions for the natural protein. The factors important to the development of such a buffer system will be discussed here.
Once a protein is liberated from its natural source by some form of cellular disruption, the protein must be solubilized in an appropriate buffer system as quickly as possible. The first treatment of the nascent buffer is either filtration or centrifugation in order to remove cellular debris and other precipitated material.
The buffer provides the proper pH environment for the protein of interest. Several factors must be taken into consideration. The pH chosen for the buffer system depends on the final use of the protein. If using the protein for some type of biological assay, e.g., an enzyme assay, the pH where the enzyme exhibits optimum activity should be chosen. When preparing a protein for a purification technique (ion exchange, gel filtration, etc.), the pH will be chosen for maximum efficacy of the purification. When no particular pH is required , the pH where the protein exhibits optimum stability is best. This is especially true for storage of the protein. Another factor to consider is the effect of temperature on buffer pH. Buffer pH changes with temperature, the magnitude of the change being dependent on the buffer itself. Finally, the buffering component of the buffer itself should not interact with the protein. For example, some enzymes are inhibited by the phosphate group of phosphate buffers. Typical buffer concentrations range from 20 to 100 mM and may require the addition of other components such as salt (150 mM for saline)for proteins sensitive to ionic strength, or metal ions such as magnesium, which is required for some enzymes to be active.
Proteins containing the amino acid, cysteine, possess a free thiol group, -SH. In the presence of other thiol groups or other cysteine amino acids in the protein chains, oxidation can occur forming a disulfide bond. For proteins that require a free thiol group (e.g., enzymes with a free thiol group at the active site), these oxidation reactions can lead to losses in biological activity. Generally speaking, proteins originally located within the cell will contain free thiol groups. Extracellular proteins generally have thiols in the form of intramolecular disulfide bonds between the cysteines of the protein. These disulfide bonds are usually crucial to tertiary structure and activity. The presence of thiols here can lead to a mixing of disulfides referred to as thiol exchange resulting in denaturation and loss of activity. When dealing with disulfide containing proteins, a reducing environment is detrimental to protein activity, therefore, low pH and free thiols are to be avoided. When working with intracellular proteins containing free thiol groups, a reducing environment that mimics the cell's interior is necessary. Addition of reagents such as dithiothreitol, reduced glutathione, or 2-mercaptoethanol to the buffer is necessary when a reducing environment is required.
Heavy metal ion contamination may be deleterious to protein activity, particularly proteins that contain free thiol groups. Aside from choosing high quality buffers and water (the major source for metal ion contamination), metal ion contamination can be eradicated by addition of ethylenediaminetetraacetic acid (EDTA) to the buffer. Typical concentrations of this metal chelating agent are 0.1 mM. Besides EDTA, sulfhydryl reagents such as those mentioned previously will also neutralize the deleterious effects of metal ions. Of course, reagents such as EDTA are avoided when protein activity demands the presence of divalent metal ions.
Temperature is an important consideration when designing a buffer system for protein stabilization. As with pH, proteins generally have several temperature optima depending on the experimental context. A good rule of thumb is proteins are more stable at reduced temperature, typically 4°C. This is especially true when proteolytic agents such as proteases are present. Although colder temperatures promote stability, they generally have a negative effect on many purification methods such as chromatography. When chromatographic efficiency is weighed against protein stability and maintenance of activity, however, activity usually wins. Unless a refolding procedure is available for the protein of interest, loss of activity cannot be tolerated during protein purification. Additionally, protein temperature optimum for biological activity is not necessarily the same as protein stability.
Aside from pH, reducing conditions, temperature, and ionic strength, some proteins require different levels of hydrophobicity for proper solvation. The requirement for buffer hydrophobicity depends on the particular protein and can vary dramatically. Typical serum proteins have excellent solubility in an aqueous salt environment and generally require no hydrophobic additives. Proteins loosely associated with membrane in vivo usually require some additive such as glycerol or sucrose for proper solubilization and activity. Typical concentrations for glycerol range from 5% to 20%. Chromatographic efficiency is reduced with increasing concentration of glycerol due to viscosity, therefore, addition of hydrophobic agents should be performed with moderation. For long term storage, buffer viscosity is not a problem. Typical glycerol concentrations can be as high as 50%.
During cellular disruption proteolytic enzymes referred to as proteases can be released into the buffer environment. These proteases then degrade the desired protein leading to protein precipitation and loss of activity. Aside from rapid purification procedures and low temperatures that reduce the protease activity, a class of compounds called protease inhibitors can be added to the buffer. These inhibitors are small molecules that interact with the active site of the protease and inhibit its proteolytic activity. Typical proteases inhibitors include diisopropylfluorophosphate (DIFP), phenylmethylsulfonyl flouride (PMSF), leupeptin, and phosphoramidon.
Caution must be exercised when handling protease inhibitors since some are very toxic. Since the addition of protease inhibitors represent an intentional contamination of the protein sample, they should not interact negatively with the protein of interest.