Expression, Purification, and Biochemical Characterization of a recombinant protein kinase (CRK1) from Leishmania mexicana

The CRKs cyclin-dependent kinases of Leishmania are essential for multiplication and survival of the parasite. Previous genetic studies have suggested CRKs as potential drug target. Further analyses were hindered by unavailability of sufficient amount of the kinase. Aim of the study is the production, purification and biochemically analysis of recombinant protein kinase (CRK1) from Leishmania mexicana. A system was developed to express and affinity-purify recombinant L. mexicana CRK1 protein from Escherichia coli, in which 6-histidine tag was added to the C-terminus of the over-expressed CRK1 kinase. Insoluble recombinant L. mexicana CRK1his was purified to nearly homogeneity using urea and was renatured by dilution and dialysis methods. Western blot analysis has confirmed the expression of the pure kinase. Work presented by this paper has confirmed the usefulness of the prokaryotic system for production of pure homogenous recombinant protein kinase of Leishmania parasite. This protein could possibly be used for inhibitory assays. Generally, same approach could be exploited for production of anti-bodies to be used in diagnosis or vaccine development.