Expression, Purification And Kinetics Of M.tuberculosis RmlA

Objective:Mycobacterium tuberculosis is the pathogen that causes tuberculosis. World health organization indicates by statistics that 9,000,000 tuberculosis occur in the world every year, and about 3,000,000 people die of tuberculosis every year. Tuberculosis has already become one of the infectious diseases which result in a high mortality. People face the challenge of multidrug-resistant strains of M. tuberculosis. It is urgent to develop the new anti-TB drugs.The cell wall is essential for mycobacterial growth, so it can be the target to develop the new drugs. The core of the cell wall consists of peptidoglycan, arabinogalactan, and mycolic acid. The mycolic acid and arabinogalactan are attached to peptidoglycan via the disaccharide linker (L-rhamnosyl-N-acetyl-glucosaminyl-phosphate). If the disaccharide linker was destroyed, the cell wall of M. tuberculosis would be destroyed and the germ would die. L-rhamnose (L-Rha) can be an important target to develop the new anti-TB drugs since it is not found in humans. Four enzymes, RmlA, RmlB, RmlC, and RmlD, involve in the synthesis of dTDP-Rha from the substrate glucose-1-phosphate. Studies of the kinetics of RmlA, RmlB, RmlC, and RmlD enzymes in-depth will be helpful to establish a perfect enzyme reaction system for screening inhibitors of RmlA enzyme to develop the anti-TB drugs.Our objectives in this study are (1) to overexpress RmlA protein by pET express vector in E.coli. (2) to purify RmlA protein by affinity chromatography. (3) to identify the purified RmlA protein by SDS-PAGE and Western blot. (4) to establish a method to detect the activity of RmlA protein by HPLC; (5) to optimize the reaction conditions of RmlA and...