The Investigation of periplasmic nitrate reductase
Thornton, Charles; Adams, Andrew K.; Thomas, John; Basu, Partha
Department of Chemistry and Biochemistry; The Project SEED Program; Duquesne University
The molybdoenzyme, periplasmic nitrate reductase (NapA), plays an important role in the vitality of the pathogenic bacterium, Campylobacter jejuni. C. jejuni is a microaerophilic bacterium that grows anaerobically by utilizing nitrate as an electron acceptor. Infection by C. jejuni is one of the most common causes of gastroenteritis in the United States. NapA cloned from C. jejuni has successfully been overexpressed in E. coli. Produced protein has also been successfully isolated and purified for further studies using a reduced methyl viologen assay. Other techniques, such as Immobilized Metal Affinity Chromatography (IMAC) and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), have been used to further study this enzyme. IMAC was performed to retain the protein in a column that contained immobilized nickel ions for the purification of the polyhistidine tag. SDS-PAGE was done to see if NapA was present in the protein sample.