32.11: Tagging and Fusion Proteins
Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and purification processes. Affinity tags, epitope tags, reporter tags, fluorescent tags, and self-splicing intein tags, are just a few of the various protein tags available.
Glutathione S-transferase Tag
Glutathione S-transferase (GST) is a 211 amino acid protein commonly employed to tag recombinant proteins. An expression vector comprising the gene of interest and the DNA sequence for GST is used for expression in a suitable host, such as E.coli. The recombinant protein can be tagged with GST at either its N-terminal or C-terminal. The GST-tag also increases the solubility of the fusion protein compared to the non-tagged native protein. Since GST is an enzyme, it has high binding specificity to its substrate, glutathione. This substrate specificity is used to purify GST-tagged proteins by affinity chromatography using a matrix of glutathione-coated beads.
Tag Cleavage and Self-splicing
While protein tags allow the target protein to be purified, they might hinder further protein analysis. In such cases, the tags are cleaved using proteolytic enzymes. Since these proteases cleave only at specific sites, fusion proteins are designed with such cleavage sites between the target protein and tag. Another method uses self-splicing protein segments, called inteins, that splice the tags from the target protein without additional enzymes. In this method, an intein segment is also recombined into the fusion protein, positioned between the tag and target protein. These inteins self-splice only under certain conditions such as the presence of thiol compounds or specific pH and temperature. Thus, splicing can be specifically induced after purification of the fusion protein to obtain the pure target protein for further analysis.
