Shikimate Kinase Expression; Towards Screening of Potential Antibacterials from Marine Sponges

Abstract

Tuberculosis is one of the world’s leading cause of mortality from a single bacterial pathogen, with over 10 million reported cases each year. There is an alarming increase in the prevalence of drug‐resistant strains, thus the need for the discovery of novel anti‐tubercular agents. The shikimate pathway is a seven‐step metabolic route that produces aromatic amino acids and other cellular metabolites. It has no mammalian counterpart, making any of the enzymes in this pathway suitable targets for screening of potential anti‐tubercular agents. Mycobacterium tuberculosis shikimate kinase (MtSK) catalyzes the 5th step of this pathway, converting shikimate to shikimate‐3‐phosphate using ATP as a co‐substrate. The overall goal of this project is to express and characterize MtSK and screen for potential anti‐tubercular agents. Transformation of XL‐1 blue competent cells was performed using a pET 21b plasmid with aroK gene inserted at the multiple cloning site. Plasmids were cloned and purified and used to transform BL 21 DE3 competent cells for subsequent protein expression. Small scale expression showed the presence of a band of increasing prominence around 20 kDa. This suggests MtSK was successfully expressed. Expression analysis for larger scale supported data from small scale. Purification, characterization, and MtSK kinetic parameters would be determined prior to enzyme inhibition studies using inhibitors like avarone (below), a marine sponge sesquiterpene quinone and derivatives thereof. Support or Funding Information East Stroudsburg University of Pennsylvania Avarone