Characterizing Tagged Cytochrome P450 Enzymes

Hello, my name is Joseph Draut, I’m a rising senior at Northwestern University studying chemical engineering. This summer, I’ve been working on a collaboration between the Tullman-Ercek lab and the Tiede lab, investigating cytochrome P450, an enzyme capable of carrying out oxygenation reactions on inactivated carbon-hydrogen bonds – a reaction that is challenging to carry out with chemical catalysts. This chemistry is particularly useful in generating industrially valuable compounds like linear alcohols, amines, or steroids. Ultimately our project seeks to investigate functionalizing P450 through modifications that would make the protein more efficient. Since P450 is relatively unstable in the cytoplasm, selectively localizing the protein to a more amenable environment may improve protein functionality. Towards this end, the Tullman-Ercek lab has added a tag (a small amino acid sequence) that drives the localization of the protein to a microcompartment – a small protein “cage” so to speak. The addition of this localizing tag alters the amino acid sequence of the protein and could therefore potentially alter the functionality. Thus, my work this summer has aimed to determine whether tagging P450 alters its functionality compared to the wildtype (untagged).

Figure 1. Cartoon depicting the localization of P450 to bacterial microcompartments after addition of a short localization sequence to the protein.

Throughout the summer, I have been able to optimize an expression strategy for P450 in E. coli and purifying using fast protein liquid chromatography (FPLC). I have further validated the activity of our tagged P450 on varied chain ethers and found it comparable to the wildtype. In the interest of using electron paramagnetic resonance (EPR) – a technique that interrogates unpaired electron systems – to further characterize our P450 variants, I quantified the robustness of P450 to freeze-thaw cycles, as EPR requires sample freezing. With these tasks done, I intend to carry out EPR based experiments in my remaining time at Argonne.

Coming from a cell-based devices background, this summer I wanted to learn about other domains of biology research and how synthetic biologists and biochemists can collaborate to apply diverse knowledge and experiences to further biological interrogation and application. I have gained a multitude of new skills and furthered my horizons in the research space, all of which I will carry with me in my future endeavors.