Production, Mutagenesis, Purification, and Kinetic Characterization of Campylobacter jejuni L-aspartate Ammonia-lyase Expressed in Escherichia coli

Authors: Zachary Kushner, Christian Nelson, Angel Alicea-Morales

SUNY Campus: SUNY Cortland

Presentation Type: Poster

Location: UU 111

Presentation #: 61

Timeslot: Session D 3:00-4:00 PM

Abstract: Campylobacter jejuni (C. jejuni) is a leading cause of bacterial gastroenteritis in humans, and exposure occurs through consumption of undercooked poultry, contact with infected animals, untreated water, and other sources. Infection commonly shows symptoms of diarrhea, cramping, abdominal pain, and fever. AspA, an enzyme involved in L-aspartate metabolism by C. jejuni, is implicated in the pathogen’s virulence. This poster will focus on the use of site directed mutagenesis to generate a panel of aspa mutants and will also focus on the introduction and expression of aspa gene in an e. coli host bacterium. The goal of this project is producing point mutations in the aspa gene, thereby altering specific amino acid residues within the AspA active site. Plasmids containing wild type or mutated aspa genes are introduced into an E. coli bacteria, which is optimized for protein production. Gene expression was induced using IPTG, followed by protein purification via fast protein liquid chromatography. Future work will include kinetic assays to characterize how these substitutions affect enzymatic activity. This work will likely result in both identification of the active site of this enzyme as well as amino acid residues important in catalysis and may lead to the development of novel anti-microbial enzyme inhibitors that provide treatment options for those infected with C. jejuni.