PHYSIOLOGY AND BIOCHEMISTRY
Optimizing conditions for active MurA purification for use in novel inhibitor studies. ANDREA
N. FROST*, BEN BOONE, AND JANUARY HAILE. Department of Biochemistry and
Molecular Biology, Centre College, Danville, KY 40422.
Antibiotic resistance necessitates the continuous development of novel inhibitors targeting a
variety of bacterial pathways. The pathway for the synthesis of peptidoglycan, a necessary
component of the bacterial cell wall, is an ideal target for antibiotic development for two reasons:
1) the cell wall is a prokaryote-specific structure, and 2) the cell wall is necessary for bacterial
survival. MurA, the target of current study, is the enzyme that catalyzes the first step in
peptidoglycan synthesis. The central goal of our project is to develop novel inhibitors that
competitively bind to the MurA catalytic sight for UDP-N-acetylglucosamine. In an effort to
study the effects of novel inhibitors on MurA, the enzyme must first be isolated and purified
using transformation, overexpression, and purification procedures. This study seeks to identify
optimal conditions for purification of active protein in high concentrations. After successful
isolation of pure MurA, conditions for maximal protein production are optimized. Isolated
protein activity is then determined through a malachite green assay. The isolation of pure MurA
through competitive ion binding columns has thus far been successful, but the enzyme has not
yet been found to be active after purification.