Characterization of Potential Antimicrobial Targets from Important Pathogens

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Authors
  1. McWilliams, M.
  2. Chan, G.
  3. Radford, C.
  4. Tokaryk, K.
  5. Russell, M.
  6. Mah, D.
Corporate Authors
Defence R&D Canada - Suffield, Ralston ALTA (CAN);Canada West Biosciences, Calgary ALTA (CAN)
Abstract
Antibiotic resistance is of increasing medical concern. As the old therapies become ineffective, new drug targets must be identified. Metabolic pathways offer attractive chemotherapeutic drug targets which have until recently been laborious to discover. The advent of large scale genome sequencing projects has revealed the information required to begin the identification and analysis of new target enzymes involved in many metabolic pathways in some of the medically important pathogens such as P. falciparum, the causative agent of malaria, and B. anthracis which causes anthrax. Results: We characterized enzymes from the following organisms: the protozoan Plasmodium falciparum, the bacilli Bacillus cereus, Bacillus anthracis, and Bacillus Subtilis, and the mycobacteria Mycobacterium tuberculosis, Mycobacterium marinum, and Mycobacterium smegmatis. To this end, we cloned the chosen enzymes into various E. coli expression vectors and induced their expression in small-scale culture. After confirmation of expression, we purified the enzymes using affinity FPLC and performed preliminary enzyme assays to determine the activity of the expressed protein. We found some of the enzymes could not be expressed at all, while others were expressed only as inclusion bodies. We performed kinetic studies on those enzymes that were expressed, and found to be active.
Keywords
Bacillus anthracis;Bacillus megaterium;Mycobacterium tuberculosis;Mycobacterium smegmatis;Plasmodium falciparum;methylthioadenosine;phosphorylase;nucleosidase;cysteine;desulfurase;expression vectors
Report Number
DRDC-SUFFIELD-CR-2005-110 — Contractor Report
Date of publication
12 Jun 2005
Number of Pages
60
DSTKIM No
CA026285
CANDIS No
524105
Format(s):
CD ROM

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