2023 100 P

Out of Nickel's Embrace: How Cation Exchange Chromatography Boosted Protein Purification Yield

By: Nam Him, Riya Maharjan, Grace Gonzales, Amir Chirar

Department: Chemistry & Biochemistry

Faculty Advisor: Dr. Misty Kuhn

Heterologous protein expression does not always produce soluble protein, which is required for downstream biochemical studies. Based on experimental results from a student research project in a Chem-343 biochemistry 1 laboratory course, we explored using cation exchange chromatography as an alternative to nickel affinity chromatography for purifying a set of predicted polyamine acetyltransferase proteins with basic isoelectric points (pIs). Therefore, we selected nine different proteins with calculated basic pIs that were as basic as or more basic than the pI of Wg-MDH and purified them using both types of chromatography to compare their purity using SDS-PAGE. During the course of the experiments, several questions arose about the accuracy of using a calculated versus experimentally determined pI for each protein, the thresholds for selecting buffer pH compared to pI, and whether the reason for failure of some proteins with nickel affinity was due to accessibility of the polyhistidine tag for purification. If the polyhistidine tag on a protein is not accessible, other positively charged regions on the protein may have the potential to interact with a cation exchange resin. Additionally, the protein could exhibit a net positive charge under specific buffer conditions, thereby facilitating its binding to the resin. We will present our current understanding of these concepts based on our experimental results thus far, since this approach has the potential to serve as a valuable purification step under these circumstances.