Leishmania mexicana: expression; characterization and activity assessment of E.coli-expressed recombinant CRK3

OBJECTIVES AND METHODS: Previous studies have shown that CRK3 protein kinase of Leishmania mexicana is a potential drug target. Therefore, the aim of this study was to provide an active protein kinase for chemical inhibitors testing. A system was developed to express and affinity-purify recombinant L. mexicana CRK3 protein from Escherichia coli. RESULTS: Biochemical analysis has confirmed the expression of the pure kinase. The bacterial-expressed kinase was found to be inactive as a monomer. The mutated CRK3-E178 protein kinase was also found to be inactive. CONCLUSION: This study suggests that cyclin binding and phosphorylation status are both important for reconstituting protein kinase activity. Work presented by this paper has confirmed the usefulness of the prokaryotic system for production of pure homogenous recombinant protein kinase of Leishmania parasite, though this system is unable to produce active CRK3 protein kinas