Exploring New Avenues for Expressing and Stabilizing Bacteriophage Polyamine Acetyltransferases for Functional Studies
Maryam Alansary, Alanis Alvarez, Julia Hun, Cristofer Leon, Hazel N. Leiva Martel
Department of Chemistry & Biochemistry
Faculty Supervisor: Misty L. Kuhn
Bacteriophages are viruses that specifically destroy bacterial cells without the use of antibiotics. The increasing prevalence of antibiotic resistant infections worldwide has driven a renewed interest in bacteriophages and their use as potential therapeutic targets. Despite this resurgence in bacteriophage research, significant gaps remain regarding our understanding of many bacteriophage-encoded proteins. For example, the biological functions and 3D protein structures of most bacteriophage proteins remain unknown. In this study, we selected six bacteriophage proteins that were previously predicted to function as polyamine acetyltransferases in order to explore their structure/function relationships. While we were able to obtain two crystal structures of two different proteins, we found that they were unstable for longer kinetic studies. Additionally, several of the proteins readily precipitated after purification, making kinetic assays and validation of functions impossible. It remains unclear whether this lack of activity reflects a true inability to acetylate polyamines, or if it is a result of protein precipitation. To address these limitations and outstanding questions, we explored alternative protein production strategies, including using cell-free protein expression and small-scale magnetic bead purification. Here, we present our initial results and avenues for further optimization.
This study was funded in part by the National Institutes of Health Department of Health and Human Services, National Institute of General Medical Sciences under Award Number R35GM133506 (to MLK).