Affinity Chromatography of Recombinant Proteins

During the last few years the use of affinity chromatography has become very widespread due to the ability, using molecular biology techniques, to modify proteins so that they can bind to specific adsorbents. The basic principle is illustrated in Figure 5. The gene encoding the protein is fused to DNA which encodes either a complete protein or a polypeptide that is to be used in the affinity process. The expressed protein is then readily purified from the host proteins by passing through the appropriate affinity adsorbent. Since only one adsorbent is needed for each particular system, one laboratory may use the same adsorbent for all its protein purifications. A brief list of some of the combinations of fusion/adsorbent is given in Table 2. These are all commercial products, and there is considerable competition, with new ones being introduced all the time. A popular term for the system is affinity tagging, with the tag being the fusion part. No one system is ideal for all proteins. In particular, the level of expression obtained can be dependent on the fusion type, and the maximum possible expression is required to optimize the overall process.

The end-product of the purification is not the original protein, but a fusion with the added protein or polypeptide. For many purposes this product is good enough, but there are methods of removing the fusion portion, generally by proteolysis. Vectors for creating fusion proteins include an amino acid sequence that is

Figure 4 Principles of affinity elution. The specifically bound protein is displaced by binding preferentially with the free ligand, W present in the elution buffer. Other proteins which may be adsorbed nonspecifically do not interact with ligand W and so remain on the column.

Figure 5 Principle of affinity tagging. The protein is expressed in recombinant form with an extra polypeptide tag. This may be a few amino acids, or may be a complete protein. The affinity adsorbent recognizes the tag, whereas untagged proteins are not adsorbed. The tag may be removed with a protease, either while still on the column, or after elution.

Figure 5 Principle of affinity tagging. The protein is expressed in recombinant form with an extra polypeptide tag. This may be a few amino acids, or may be a complete protein. The affinity adsorbent recognizes the tag, whereas untagged proteins are not adsorbed. The tag may be removed with a protease, either while still on the column, or after elution.

recognized by a highly selective proteolytic enzyme such as a blood-clotting factor. Treatment with this enzyme, either before or after the fusion protein has been eluted from the affinity column, releases the original protein, though still with a few extra amino acids in most cases.

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Solar Panel Basics

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