Expression, Purification, and Protein Crystallography Trials in Influenza Polymerase Acidic Protein Fusion
Kyle Benjamin Harris
Influenza viruses are responsible for annual epidemics, as well as previous pandemics including the H1N1 pandemic in 2009. In any given year, worldwide deaths due to influenza virus can reach 650,000. Due to influenza’s continued threat to global health, along with increasing drug resistance, new antiviral medications are imperative to safeguarding the global community. An attractive target of novel antiviral drugs is the Polymerase Acidic Protein (PA) of influenza. PA is essential to influenza replication, and its amino acid composition is highly conserved among fluA, fluB, and fluC strains. Currently, crystal forms of PA do exist, but these forms do not provide an accessible binding pocket for inhibition. This inaccessibility stimulates the need for additional crystal forms that could result in structure-based synthesis of novel inhibitory compounds. In order to initiate crystal trials, large scale expression and purification of PA must take place. Expression of recombinant proteins in Escherichia coli bacteria cells often result in decreased production yields due to insolubility and inclusion body formation. To combat these effects, a PA construct was created in fusion with Green Fluorescent Protein (GFP). As well, the lysis buffer in the purification phase was optimized using solubilizing additives. Purification of the expressed construct was done through Immobilized Metal Chromatography, using a Nickel column, and Size Exclusion Chromatography, respectively. Purified samples underwent a Precrystallization Test, where results from this test were used to determine protein concentration used in large-scale sparse matrix crystallography screens, totaling 192 conditions. Two conditions from these screens gave interesting results and are under investigation.
Douglas S. Daniels
Primary Advisor's Department
Stander Symposium project, College of Arts and Sciences
United Nations Sustainable Development Goals
Good Health and Well-Being
"Expression, Purification, and Protein Crystallography Trials in Influenza Polymerase Acidic Protein Fusion" (2022). Stander Symposium Projects. 2453.