The Arginine-Phenylglyoxal Peptide Tag (APP-tag) – A Ubiquitous Strategy for Protein Bioconjugation In Vivo

by Olivia Steiner

mentor: Rebecca Scheck, Chemistry; funding source: Usdin Summer Scholars Fund

Our bodies are composed of millions of cells and within them are proteins that work to maintain nearly all life-sustaining processes. Proteins are the macromolecules behind the immune response, oxygen transport, reproduction, food metabolism, and much more. Since proteins are essential to life, it is important to gain an in-depth understanding of their functions and the roles they play in different biochemical pathways in the body.

There are a plethora of proteins that make up an organism and if one protein is not synthesized correctly, or even misfolded, this often results in disease. If researchers learn more about proteins and their functions, they can apply their knowledge to ultimately fix the protein, or reverse the damage it caused, when something goes wrong. It is clear that it is important for scientists to have methods to study proteins, however, there are few simple, universal strategies that exist to examine them.

The most common way that researchers study proteins is by attaching fluorescent molecules to proteins that allow them to be easily visualized. Although this may sound straightforward, in practice, there are many challenges that accompany attaching a molecule to a protein in living cells. For starters, the chemicals used need to be able to get through the cell membrane, and the chemical reaction taking place must not interfere with any of the other thousands of chemical reactions taking place in the cell. Additionally, the chemicals used cannot be toxic or lead to toxic byproducts that would kill or harm the cell. Lastly, proteins are made up of twenty different amino acids and each amino acid has a different chemical functionality, which means the chemistry employed to attach a molecule to a protein must only be compatible with one of those twenty amino acids. The attachment of a molecule to a protein is coined protein bioconjugation, and it clearly has many challenges. As a result, I have spent the past year in the Scheck Lab at Tufts working on a ubiquitous strategy for protein bioconjugation and through the Tufts Summer Scholars Program, I was able to spend the summer examining different strategies for protein bioconjugation by drafting a literature review.

I found that protein bioconjugation strategies essentially fall into three categories that all have their own advantages and disadvantages. Researchers either use chemicals to modify naturally occurring amino acids, enzymes, which are another type of protein, to modify naturally occurring amino acids, or they engineer an unnatural amino acid into a protein and use chemistry to modify that unnatural amino acid. Collectively, I found that these methods seem to have two main, overarching limitations. The first limitation is that the strategies are not universal and have to be adapted to the protein being studied and the second is that many aren’t applicable in vivo, or in living cells. Researching disparate strategies for bioconjugation this summer shed light on the reasons my project in the Scheck Lab would be a novel method for protein bioconjugation and ultimately protein tagging. My project in the Scheck Lab is to develop a tag that could be incorporated into any protein of interest and it would take advantage of chemistry that is compatible with living cells. The tag would allow researchers to attach a diverse array of molecules to any protein of interest. In conclusion, it is essential to develop protein bioconjugation strategies that could be utilized to study proteins in live cells and I spent the summer exploring the current strategies available for protein bioconjugation.