Marcin Ogrodniczuk (East Stroudsburg University )| René Fuanta (East Stroudsburg University )
Tuberculosis is a respiratory infection with over 10 million reported cases each year. This infection rate is responsible for over two million deaths annually, second only to HIV in fatalities among infectious diseases. Since 1960 tuberculosis rates have steadily declined worldwide as medical technology has advanced to produce more effective antibiotics. However, recent studies have shown that tuberculosis rates have seen a steady increase. This observation suggests that there is an alarming increase in the prevalence of drug‐resistant strains of tuberculosis, thus the need for the discovery of novel anti‐tubercular agents. Targeting potential enzymatic pathways for drug discovery requires the pathway to possess minimal overlap with the host. The shikimate pathway is a seven‐step metabolic route that produces aromatic amino acids and other cellular metabolites. This pathway is typically present in microorganisms and has no mammalian counterpart, making any of the enzymes in this pathway suitable targets for screening of potential anti‐tubercular agents. The target enzyme here, Mycobacterium tuberculosis Shikimate Kinase (MtSK), catalyzes the 5th step of this pathway, converting shikimate to shikimate‐3‐phosphate. The overall goal of this project is to express and characterize MtSK in order to screen for potential anti‐tubercular agents. Initial methods included a bacterial transformation of XL‐1 blue competent E.coli cells. This preliminary transformation was performed using a pET-21b plasmid with an aroK gene inserted at the multiple cloning site. Successfully transformed XL-1 blue cells were cloned and a second transformation using BL21 DE3 competent cells as an expression host was performed. Small scale expression showed the presence of a band around 20 kDa. The theoretical mass of the enzyme is 19.6 kDa which suggests MtSK was successfully expressed within the transformed cells. Expression analysis for large-scale supported data from small scale as a band of 20 kDa once again appeared in the verification SDS-PAGE gel. Purification of MtSK was carried out through nickel affinity chromatography. Subsequently, kinetic characterization and inhibitors studies will be performed using inhibitors like avarone and hymenidin.