Closing Comments

This summer, I was fortunate to work with a team in Dr. Allison’s lab on a project characterizing importin binding to thyroid hormone receptor alpha. Importins are proteins that play important roles in transporting molecules into the nucleus of the cell; by binding to a target protein, and then binding to nuclear pore complex on the nuclear membrane, importins can translocate and release cargo across the nuclear membrane. Thyroid hormone receptor alpha (TRα) is a transcription factor; that is, it is a protein that regulates gene expression, in this case in response to the presence of thyroid hormone, or T3. The receptor shuttles back and forth between the nucleus, but the precise methods of localization of TRα have yet to be fully characterized.

In order to study the interaction between various importins and TRα, we focused on generating fusion proteins – the full TRα protein or pieces of TRα with a peptide tag added to it. These tags will help both in future experiments as well as in protein purification. In order to generate these, we had to insert the gene encoding TRα into a plasmid, or small, circular piece of DNA. This plasmid could then be transformed into bacteria; bacteria would replicate the plasmid as they reproduced. This allowed us to collect a great deal of our gene. We could then move our insert (still in the correct orientation) into a new plasmid, one that encoded the peptide tag as well, and transform this into bacteria. These bacteria would multiply, generating large amounts of or plasmid and insert; this could then be transformed into bacteria more ideal for expressing our protein. From these cells, we could purify our protein and obtain it for future experiments.

Though we ran into some issues with protein expression, we have made progress, and are on our way to making more this semester. However, I feel that working in a team to solve the issues we have had with protein expression have made me more prepared to deal with setbacks in the future. Having to think about, research, and discuss options of how to proceed has given me, I think, a better picture of research as a process.

Even though I was unable to work on my original project this summer, I feel very fortunate to have worked on this project, and I am excited to move forward with it as we move into the fall semester.