2023 99 P

Optimizing Soluble Protein Expression of a Gcn5-related N-acetyltransferase through C-Terminal Truncations

By: Joshita Kamalakannan, Daniel Figueroa Paniagua

Department: Chemistry & Biochemistry

Faculty Advisor: Dr. Misty Kuhn

Our laboratory is trying to identify ways to understand what conditions help solubly express challenging proteins that belong to the Gcn5-related N-acetyltransferase superfamily. Some proteins we have tested are not soluble using standard expression conditions. Therefore, we are using truncations as a salvage strategy to determine if removing a portion of the protein C-terminus will improve solubility. When we comparted a set of five GNAT proteins with C-terminal extensions we found these proteins have a conserved amino acid linker connecting the main body of the protein to a predicted C-terminal alpha helix. Prior studies have shown that truncating proteins at this conserved region has improved soluble expression. However, it is not known whether the linker is required for soluble expression or if it can be removed. Therefore, we designed four distinct truncation constructs at this conserved amino acid region. Once the correct sequence was confirmed, all four truncated proteins were expressed using standard conditions and later purified using a nickel-affinity column on an FPLC. We determined that these new truncations increased the amount of soluble protein compared to the WT non-truncated protein. This is crucial to understanding the role of what components of GNATs can be altered to obtain pure protein for enzyme kinetics studies.