Modeling a Series of GNAT proteins with N-terminal Polyhistidine Tags and Their Effects on Protein Expression and Purification
By: Phelan Glenn
Department: Chemistry & Biochemistry
Faculty Advisor: Dr. Misty Kuhn
Our lab has been working toward understanding how the molecular structure of enzymes that belong to the GNAT superfamily affect protein function. We are currently investigating a diverse set of proteins that are predicted to acetylate polyamines. Since none of the structures of these proteins are known, we generated alpha2fold models to compare their structures and active sites. Collectively, our laboratory has also been working to purify each of these enzymes for enzyme kinetic studies. However, many of the proteins are not expressed or produce insoluble protein. In some cases, the protein is expressed but is not able to be purified. We hypothesized that for some of these proteins the poly-histidine tag attached to the N-terminus of the protein may be inaccessible by interacting with the active site. Therefore, we generated models of these proteins with and without the tag, examined whether the tag was observed in the active site, and compared the modeling results with the in vitro protein purification results. This information will be used to select proteins that would be good candidates for changing the location of the polyhistidine tag as an alternative strategy for producing pure protein.