Protein tags

The project I worked on this summer is ongoing, so by the time I joined, a lot of work had already been completed. The first “phase” of our project involves creating a plasmid (or small, circular piece of DNA) that when expressed will yield our fusion protein (TRα or individual domains with a peptide tag fused to it).

But what are those peptide tags, and what is the point of them? To be honest, I’m still learning about them myself. In an effort to keep track of the entire project – it was very easy to get lost in the details of what we were doing, and lose sight of our eventual goal – I attempted to learn more about the tags we were using: namely, GST and poly-His tags.

GST stands for glutathione-S-transferase. They are a family of enzymes which play a variety of roles in the cell, the most well-known being their anti-toxic properties. After expressing the protein in a bacterial cell, the tag will allow us to easily extract it. In a procedure called a pull down assay, beads coated in glutathione are added to the cell protein mixture; the GST tagged protein will “stick” to the beads, allowing for easy extraction.  

The “his” tag is actually a 6-histidine tag. Histidine is an amino acid, and the tag is simply six of these amino acids added to the protein. This tag has the benefit of being small and not interfering too much with the functionality and expression of the protein. His-tagged proteins can be purified using a resin that contains bound metal ions, such as nickel; the his-tag has an affinity for the metal ions and binds to them, allowing for purification. His tags can also be used for studying protein-protein interactions; for example, the interaction between TRα and importins/exportins.

My next entry will be about DNA sequencing and the issues we’ve been facing with it this summer.