Cloning of enolase gene without intron, expression, purification and kinetic characterization of the enzyme from Theileria annulata

Tropical theileriosis is caused by infection with the apicomplexan parasite Theileria annulata that is spread by ticks of the genus Hyalomma and high rate morbidity and mortality in high efficiency cattle. Thus, tropical theileriosis is a common and important disease causing huge economic losses. In recent years, parasite resistance has been reported to most effective antitheilerial drug used for the treatment of this disease. This situation has given rise to the need for alternative methods of treatment. Glycolysis play crucial roles in the ATP supply, important for the viability and virulence of Theileria annulata. This pathway is, therefore, candidate targets for antitheilerial drugs. Enolase is a key glycolytic enzyme that catalyzes the inter conversion of 2-phosphoglycericacid (2-PGA) and phosphoenolpyruvate (PEP). Also, recent reports described that enolase expressed at the cell surface of several pathogenic organisms. The surface enolase acts as a plasminogen-binding receptor and plays an important role in tissue invasion. Since enolase is a key glycolytic enzyme and plasminogen-binding receptor for Theileria annulata, enolase can be selected as a macromolecular target of therapy of tropical theileriosis. In this study, an intron sequence present in Theileria annulata enolase enzyme gene was removed by PCR site directed mutagenesis and the gene was cloned into pGEM-T Easy vector. Removal of intron sequence was confirmed by DNA sequencing. The gene was than subcloned into pLATE31 vector, having an C-terminal 6xHistaged sequence, and exspressed in Escherichia coli BL21(DE3) cells. The enzyme was purified by affinity choromatography using Ni-NTA agarose coloumn. Staedy state kinetic parameters of the enzyme was determined using GraFit 3.0. Because of lack of RNA sample from Theileria annulata, a strategy has been design by using PCR site directed mutagenesis for removal of an intron sequence from a gene with the ultimate aim of producing active enzyme that will be available for structure based drug design studies. To our knowledge, present study is first to be applied with the aim of production of active protein from a gene that originally had an intron sequence. Expression and purification of active enzyme is of importance for novel drug design research and strategies to overcome the diseases. High quantities (30 mg per liter culture) of pure recombinant Theileria annulata enolase has been obtained in a higly purified form (> 95 %) following intron removal. Molecular weight of purified protein gave a single band on SDS-PAGE at about 48 kDa that is in aggrement with the mass calculated from the amino acid sequence. Enzyme kinetic measurements using 2-PGA as substrate gave a specific activity of 43 U/mg, Km=106 µM, Vmax=0,132, kcat=37 s-1 and kcat/Km=3,5x105 M-1.s-1. These values have been determined for the first time from this parasite and availability of large quantities of enolase enzyme will facilitate further kinetic and structural characterization towards design of new antitheilerial drugs.