Section 3.1.5.2. Affinity purification

The most powerful purification steps are those that most clearly differentiate the desired protein from the other proteins present. Many proteins bind specifically to substrates, products and/or other proteins. In some cases, it is possible to use specific ligands to design columns to which the desired protein will bind selectively. For example, it may be possible to chemically link the substrate or product of a particular enzyme to an inert support. If the modification to the small molecule needed to link it to the support is chosen so that it does not interfere with the binding of the enzyme, the modified resin can be used to purify the protein by affinity chromatography. If, as expected, the desired protein binds selectively, it can usually be eluted by washing the column with the same substrate used to prepare the column. This is a powerful procedure and can produce greater than 100-fold purification in a single step. Although this is a fairly well developed field, and there is sufficient experience to show that the process is often fruitful, it must be said that the development of an efficient and effective affinity column and an attendant purification procedure can be long, difficult and, depending on the ligand and/or activated resin, sometimes expensive. In addition, the preparation of the column usually involves some moderately sophisticated chemistry; if such a step is contemplated, it is helpful to have the requisite chemical sophistication.

Immuno-affinity chromatography is a classic affinity method that uses affinity media created by coupling antibodies (either monoclonal or polyclonal) specific for the protein of interest to an activated resin. Theoretically, if good antibodies are available in sufficient quantity, this should be a powerful and widely applicable method. However, immuno-affinity chromatography has two severe limitations. In most cases, the interaction between the antibody and antigen is so tight that harsh conditions are necessary to elute the bound protein, potentially resulting in denaturation of the protein. Additionally, scaling up the procedure for isolation of 5–10 mg of protein is usually not feasible because of the large quantities of antibody required for column preparation.

Because the process of affinity chromatography is so powerful, and the development of a specific affinity column is difficult, considerable effort has been expended on the development of general procedures for affinity chromatography. As discussed previously, it is possible to modify the recombinant protein so that it contains a sequence element that can be used for affinity chromatography. Numerous systems are being marketed that pair vectors for creation of fusion proteins with appropriate resins for affinity purification. Examples of these fusion element–affinity resin pairs include His₆–Ni²⁺-nitrilotriacetic acid, biotinylation-based epitopes–avidin, calmodulin-binding peptide–calmodulin, cellulose or chitin-binding domains–cellulose or chitin, glutathione S-transferase–glutathione, maltose-binding domain–amylose, protein A domains–IgG, ribonuclease A S-peptide–S-protein, streptavidin-binding peptides–streptavidin and thioredoxin–phenylarsine oxide.

Several considerations are important in choosing a strategy for expression and purification of a fusion protein. Some of these issues have already been discussed (see Section 3.1.3.3). The most fundamental, and unfortunately least predictable, is what construct will produce large amounts of the recombinant protein. The presence of fusion proteins and/or purification tags perturbs the recombinant protein to a greater or lesser degree. Perturbation can in some cases be beneficial, with the fusion protein aiding in vivo folding or in vitro refolding. There is also the issue of whether or not to remove the tag or fusion protein. Removal of the tag usually involves engineering a site for a specific protease, digestion with that protease and subsequent purification to isolate the final cleaved product. Additional issues should also be addressed. Most of the well developed systems allow for the elution of the fusion protein from the affinity resin under relatively mild conditions that should not harm most proteins. However, the method of elution should be considered with respect to the specific requirements of the protein of interest. Since the costs of using the different systems on a large scale varies significantly, it is wise to calculate the expense associated with scaling up, allowing for the cost and lifetime of the affinity resin, the cost of the reagent used for elution and the cost of the protease if the tag is to be removed. Finally, the nature of the fusion element–affinity resin interaction should be considered. Some of these systems, such as the His₆ tag, can be used for purification under denaturing conditions, which is a considerable advantage if the desired recombinant protein is found in inclusion bodies.