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.
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Acknowledgments
We thank Dr. Castillo from Proquimur Company for allowing us the nanoLC/MS in collaboration of Agilent Technologies Company in Germany. DNA sequencing was carried out in “Unidad de Biología Molecular y Análisis Genético” of Servicios Técnicos de Investigación (Universidad de Alicante). Financial support is gratefully acknowledged from MCYT (BIO2002-03179).
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Communicated by K. Horikoshi
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Díaz, S., Pérez-Pomares, F., Pire, C. et al. Gene cloning, heterologous overexpression and optimized refolding of the NAD-glutamate dehydrogenase from Haloferax mediterranei. Extremophiles 10, 105–115 (2006). https://doi.org/10.1007/s00792-005-0478-8
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DOI: https://doi.org/10.1007/s00792-005-0478-8