Figure 8–10 Protein purification by chromatography. Typical results obtained when three different chromatographic steps are used in succession to purify a protein. In this example, a homogenate of cells was first fractionated by allowing it to percolate through an ion-exchange resin packed into a column (A). The column was washed to remove all unbound contaminants, and the bound proteins were then eluted by pouring a solution containing a gradually increasing concentration of salt onto the top of the column. Proteins with the lowest affinity for the ion-exchange resin passed directly through the column and were collected in the earliest fractions eluted from the bottom of the column. The remaining proteins were eluted in sequence according to their affinity for the resin—those proteins binding most tightly to the resin requiring the highest concentration of salt to remove them. The protein of interest was eluted in several fractions and was detected by its enzymatic activity. The fractions with activity were pooled and then applied to a gel-filtration column (B). The elution position of the still-impure protein was again determined by its enzymatic activity, and the active fractions were pooled and purified to homogeneity on an affinity column (C) that contained an immobilized substrate of the enzyme.
gene for protein of interest rapid purifcation of tagged protein and any associated proteins
Figure 8–11 epitope tagging for the purification of proteins. Using standard genetic engineering techniques, a short peptide tag can be added to a protein of interest. If the tag is itself an antigenic determinant, or epitope, it can be targeted by an appropriate antibody, which can be used to purify the protein by immunoprecipitation or affinity chromatography.
In other cases, an entire protein is used as the recognition tag. When cells are engineered to synthesize the small enzyme glutathione S-transferase (GST) attached to a protein of interest, the resulting fusion protein can be purified from the other contents of the cell with an affinity column containing glutathione, a substrate molecule that binds specifically and tightly to GST.
As a further refinement of purification methods using recognition tags, an amino acid sequence that forms a cleavage site for a highly specific proteolytic enzyme can be engineered between the protein of choice and the recognition tag. Because the amino acid sequences at the cleavage site are very rarely found by chance in proteins, the tag can later be cleaved off without destroying the purified protein.
This type of specific cleavage is used in an especially powerful purification methodology known as tandem affinity purification tagging (TAP-tagging). Here, one end of a protein is engineered to contain two recognition tags that are separated by a protease cleavage site. The tag on the very end of the construct is chosen to bind irreversibly to an affinity column, allowing the column to be washed extensively to remove all contaminating proteins. Protease cleavage then releases the protein, which is then further purified using the second tag. Because this two-step strategy provides an especially high degree of protein purification with relatively little effort, it is used extensively in cell biology. Thus, for example, a set of approximately 6000 yeast strains, each with a different gene fused to DNA that encodes a TAP-tag, has been constructed to allow any yeast protein to be rapidly purified.
Purified Cell-free Systems Are Required for the Precise Dissection of Molecular Functions
Purified cell-free systems provide a means of studying biological processes free from all of the complex side reactions that occur in a living cell. To make this possible, cell homogenates are fractionated with the aim of purifying each of the individual macromolecules that are needed to catalyze a biological process of interest. For example, the experiments to decipher the mechanisms of protein synthesis began with a cell homogenate that could translate RNA molecules to produce proteins. Fractionation of this homogenate, step by step, produced in turn the ribosomes, tRNAs, and various enzymes that together constitute the protein-synthetic machinery. Once individual pure components were available, each could be added or withheld separately to define its exact role in the overall process.