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.

Immunolocation and enzyme activity analysis of Cryptosporidium parvum enolase

“…In this study, the purified rCpEno could convert 2-PGA to PEP and the reaction activity was 33.5 U/mg, which was similar with P. falciparum (30 ± 3 U/mg) and T. annulata (40 U/mg) [59, 60]. Compared with other apicomplexan parasites, the K m value of rCpEno (0.571 mM) was higher than in P. falciparum (0.041 ± 0.004 mM), T. gondii ENO1 (0.0768 ± 0.0064 mM), T. gondii ENO2 (0.0777 ± 0.0049 mM) and T. annulata (0.106 mM) [57, 59, 60].…”

“…[37, 43, 55], T. annulata [59], and was in contrast to studies of T. gondii enolase which reported two stage-specific glycolytic enolases, ENO1 and ENO2 [20, 56, 57]. …”

“…Compared with other apicomplexan parasites, the K m value of rCpEno (0.571 mM) was higher than in P. falciparum (0.041 ± 0.004 mM), T. gondii ENO1 (0.0768 ± 0.0064 mM), T. gondii ENO2 (0.0777 ± 0.0049 mM) and T. annulata (0.106 mM) [57, 59, 60]. …”

“…C. parvum 106 EYGFT 110 , C. muris 103 EYGYN 107 , E. tenella 103 EWGWS 107 , N. caninum 103 EWGYS 107 , P. falciparum 104 EWGWS 108 , T. annulata 103 EWGYC 107 , T. gondii ENO1 103 EWGYS 107 and T. gondii ENO2 103 EWGWC 107 ) [31, 55–59], which is more closely related to plant enolase (i.e. A. thaliana 103 EWGYC 107 ) than to the mammalian counterpart.…”

Molecular characterization of enolase gene from Taenia multiceps

“…Enolase is a multifunctional protein that is characterized as catalyzing the reversible dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate (PEP) and plasminogen receptor on the surface of several pathogens (Cayir et al, 2014;Ramajo-Hernández et al, 2007). Furthermore, the protein could also induce host-protective immunity and is considered potential vaccine candidate that has been evaluated in several helminths (Yang et al, 2010;Chen et al, 2012).…”