Cloning, sequencing and functional expression of cytosolic malate dehydrogenase from Taenia solium: Purification and characterization of the recombinant enzyme
Por:
Nava G., Laclette J.P., Bobes R., Carrero J.C., Reyes-Vivas H., Enriquez-Flores S., Mendoza-Hernández G., Plancarte A.
Publicada:
1 jul 2011
Resumen:
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 36517 Da. 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 409 U/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 962 s(-1), respectively. Additionally, a partial characterization of TsMDH gene structure after analysis of a 1.56 Kb
Filiaciones:
Nava G.:
Dept Microbiol & Parasitol, Mexico City, DF, Mexico
Univ Nacl Autonoma Mexico, Fac Med, Mexico City 04510, DF, Mexico
Laclette J.P.:
Univ Nacl Autonoma Mexico, Inst Invest Biomed, Dept Inmunol, Mexico City 04510, DF, Mexico
Bobes R.:
Univ Nacl Autonoma Mexico, Inst Invest Biomed, Dept Inmunol, Mexico City 04510, DF, Mexico
Carrero J.C.:
Univ Nacl Autonoma Mexico, Inst Invest Biomed, Dept Inmunol, Mexico City 04510, DF, Mexico
Reyes-Vivas H.:
Laboratorio de Bioquímica-Genética y División de Investigación, Instituto Nacional de Pediatría 04530 México, DF., Mexico
Enriquez-Flores S.:
Laboratorio de Bioquímica-Genética y División de Investigación, Instituto Nacional de Pediatría 04530 México, DF., Mexico
Mendoza-Hernández G.:
Univ Nacl Autonoma Mexico, Fac Med, Dept Bioquim, Mexico City 04510, DF, Mexico
Plancarte A.:
Dept Microbiol & Parasitol, Mexico City, DF, Mexico
Univ Nacl Autonoma Mexico, Fac Med, Mexico City 04510, DF, Mexico
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