title: Gene cloning, heterologous overexpression and optimized refolding of the NAD-glutamate dehydrogenase from Haloferax mediterranei.
abstract: The NAD-dependent glutamate dehydrogenase (GDH) gene from the halophilic archaeon Haloferax mediterranei has been cloned. The analysis of the nucleotide sequence revealed an open reading frame of 1323 bp that encodes a NAD-GDH. The amino acid sequence displayed high homology with those from other sources, especially the highly conserved residues involved in 2-oxoglutarate binding. The expression of this gene in Escherichia coli, the refolding and further characterization, yielded a fully active NAD-GDH with the same features than those found for the wild-type enzyme. This halophilic NAD-GDH showed a highly dependence on salts for both stability and activity, being essential for the refolding of the recombinant enzyme.
authors.affiliation: División de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Alicante, Spain.
authors.forename: Susana
authors.initials: S
authors.lastname: Díaz
authors.forename: Francisco
authors.initials: F
authors.lastname: Pérez-Pomares
authors.forename: Carmen
authors.initials: C
authors.lastname: Pire
authors.forename: Juan
authors.initials: J
authors.lastname: Ferrer
authors.forename: María-José
authors.initials: MJ
authors.lastname: Bonete
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D015723:Gene Library; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019623:Haloferax mediterranei / Q000201:enzymology* / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011994:Recombinant Proteins; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D019843:Archaeal Proteins; D011994:Recombinant Proteins; D005969:Glutamate Dehydrogenase