Truman College1, Biology, Chicago, IL 60640 Hope College2, Biology, Holland, MI 49423
In known sequenced bacterial genomes, nfoD is a gene that codes for a step in the histidine degradation pathway, which converts N-Formyl-L-Glutamate to L-Glutamate and Formate. It is essential for these bacteria to synthesize glutamate in order for them to uptake inorganic nitrogen into the cell. Mycobacterium smegmatis and Streptomyces coelicolor do not have the nfoD gene, but are known to catabolize histidine. An alternative gene in both organisms, nfoD2, is hypothesized to serve the same function as nfoD, though they share no sequence similarities. nfoD2 was amplified from both organisms using PCR, and cloned into the expression vector, pDEST-His-MBP, then transformed into E. coli BL21 Codon Plus-RIL cells using Gateway Cloning technology. The gene was overexpressed in these cells, then the protein was purified using affinity chromatography and analyzed by SDS-PAGE. The purified protein will be used in biochemical assays to validate the hypothesized gene function.