Purifying an Intrinsically Disordered Plant Regulatory Protein

Renee in lab

Funding Source

Students

2019
College

Faculty

Professor of Biology

Project Summary

This past summer I was funded through the Jumpstart for Juniors grant to independently explore a research project introduced to me the previous semester in BIOL 425. This project focused on an unknown protein named PIR implicated in transcriptional control of the model plant system Arabidopsis thaliana. Preliminary data by the Wagner group had shown that PIR could be involved in regulation of drought stress response, making PIR potentially relevant to the agricultural industry. Moreover, PIR is predicted to be intrinsically disordered, meaning it has no set structure like many other proteins such as enzymes. Intrinsically disordered proteins are a hot topic in biology right now because many can phase separate, forming membraneless organelles that can organize protein/RNA complex components or sequester important regulatory factors. In order to see if PIR may also demonstrate this novel regulatory mechanism, I wanted to study its biophysical properties.

I was given the freedom in this project to independently design experiments and develop my molecular biology skillset. Throughout the summer, I optimized the induction time for PIR expression in e. coli, then tested various purification methods such as His-tag and FLAG tag affinity chromatography, anion exchange chromatography, and an unusual boiling purification method. Likewise, I learned to clone and began to develop biologically interesting PIR mutants, such as a version mimicking the phosphorylated form. Although I was not able to obtain sufficiently pure PIR in order to conduct phase separation assays or probe its secondary structure through spectroscopy methods such as circular dichroism, I was able to learn a variety of techniques and learn how to efficiently and independently conduct research. I will continue to apply the skills I have learned throughout my senior year as I continue my involvement with the project and the Wagner lab.