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 pro - vide an active protein kinase for chemical in - hibitors testing. A system was developed to ex - press 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 im - portant for reconstituting protein kinase activity. Work presented by this paper has confirmed the usefulness of the prokaryotic system for produc - tion of pure homogenous recombinant protein ki - nase of Leishmania parasite, though this system is unable to produce active CRK3 protein kinase.