title: Cloning of intron-removed enolase gene and expression, purification, kinetic characterization of the enzyme from Theileria annulata.
abstract: Tropical theileriosis is a disease caused by infection with an apicomplexan parasite, Theileria annulata, and giving rise to huge economic losses. In recent years, parasite resistance has been reported against the most effective antitheilerial drug used for the treatment of this disease. This emphasizes the need for alternative methods of treatment. Enolase is a key glycolytic enzyme and can be selected as a macromolecular target of therapy of tropical theileriosis. In this study, an intron sequence present in T. annulata enolase gene was removed by PCR-directed mutagenesis, and the gene was first cloned into pGEM-T Easy vector and then subcloned into pLATE31 vector, and expressed in Escherichia coli cells. The enzyme was purified by affinity chromatography using Ni-NTA agarose column. Steady-state kinetic parameters of the enzyme were determined using GraFit 3.0. High quantities (~65 mg/l of culture) of pure recombinant T. annulata enolase have been obtained in a higly purified form (>95 %). Homodimer form of purified protein was determined from the molecular weights obtained from a single band on SDS-PAGE (48 kDa) and from size exclusion chromatography (93 kDa). Enzyme kinetic measurements using 2-PGA as substrate gave a specific activity of ~40 U/mg, K m: 106 μM, kcat: 37 s(-1), and k cat/K m: 3.5 × 10(5) M(-1) s(-1). These values have been determined for the first time from this parasite enzyme, and availability of large quantities of enolase enzyme will facilitate further kinetic and structural characterization toward design of new antitheilerial drugs.
authors.affiliation: Department of Bioengineering, Faculty of Engineering and Natural Sciences, Gaziosmanpasa University, 60240, Tokat, Turkey.
authors.forename: Ebru
authors.initials: E
authors.lastname: Cayir
authors.forename: Aysegul
authors.initials: A
authors.lastname: Erdemir
authors.forename: Ebru
authors.initials: E
authors.lastname: Ozkan
authors.forename: Murat
authors.initials: M
authors.lastname: Topuzogullari
authors.forename: Zeynep Busra
authors.initials: ZB
authors.lastname: Bolat
authors.forename: Ayberk
authors.initials: A
authors.lastname: Akat
authors.forename: Dilek
authors.initials: D
authors.lastname: Turgut-Balik
mesh_terms: D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology; D001483:Base Sequence; D001709:Biotechnology; D002417:Cattle; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics; D015195:Drug Design; D017125:Genes, Protozoan; D007438:Introns; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010751:Phosphopyruvate Hydratase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D016796:Theileria annulata / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D013801:Theileriasis / Q000188:drug therapy / Q000469:parasitology
chemical_list: D000981:Antiprotozoal Agents; D016054:DNA, Protozoan; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D010751:Phosphopyruvate Hydratase