title: Cloning, sequencing and functional expression of cytosolic malate dehydrogenase from Taenia solium: Purification and characterization of the recombinant enzyme.
abstract: We report herein the complete coding sequence of a Taenia solium cytosolic malate dehydrogenase (TscMDH). The cDNA fragment, identified from the T. solium genome project database, encodes a protein of 332 amino acid residues with an estimated molecular weight of 36517Da. For recombinant expression, the full length coding sequence was cloned into pET23a. After successful expression and enzyme purification, isoelectrofocusing gel electrophoresis allowed to confirm the calculated pI value at 8.1, as deduced from the amino acid sequence. The recombinant protein (r-TscMDH) showed MDH activity of 409U/mg in the reduction of oxaloacetate, with neither lactate dehydrogenase activity nor NADPH selectivity. Optimum pH for enzyme activity was 7.6 for oxaloacetate reduction and 9.6 for malate oxidation. K(cat) values for oxaloacetate, malate, NAD, and NADH were 665, 47, 385, and 962s(-1), respectively. Additionally, a partial characterization of TsMDH gene structure after analysis of a 1.56Kb genomic contig assembly is also reported.
authors: Departamento de Microbiología y Parasitología y, Universidad Nacional Autónoma de Mexico, Facultad de Medicina, D.F., Mexico.
authors: Gabriela
authors: G
authors: Nava
authors: Juan P
authors: JP
authors: Laclette
authors: Raúl
authors: R
authors: Bobes
authors: Julio C
authors: JC
authors: Carrero
authors: Horacio
authors: H
authors: Reyes-Vivas
authors: Sergio
authors: S
authors: Enriquez-Flores
authors: Guillermo
authors: G
authors: Mendoza-Hernández
authors: Agustín
authors: A
authors: Plancarte
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D018076:DNA, Complementary / Q000737:chemistry; D004795:Enzyme Stability; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis; D041201:Taenia solium / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; D009243:NAD; D008291:Malate Dehydrogenase