Open AccessNADP‐dependent isocitrate dehydrogenase from the halophilic archaeon Haloferax volcanii : cloning, sequence determination and overexpression in Escherichia coli
Author(s) -
Camacho Mónica,
Rodrı́guezArnedo Adoración,
Bonete Marı́aJosé
Publication year - 2002
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2002.tb11125.x
Subject(s) - haloferax volcanii , isocitrate dehydrogenase , biochemistry , biology , escherichia coli , microbiology and biotechnology , peptide sequence , amino acid , nucleic acid sequence , enzyme , gene , archaea
A gene encoding NADP‐dependent Ds‐ threo ‐isocitrate dehydrogenase was isolated from Haloferax volcanii genomic DNA by using a combination of polymerase chain reaction and screening of a λ EMBL3 library. Analysis of the nucleotide sequence revealed an open reading frame of 1260 bp encoding a protein of 419 amino acids with 45 837 Da molecular mass. This sequence is highly similar to previously sequenced isocitrate dehydrogenases. In the alignment of the amino acid sequences with those from several archaeal and mesophilic NADP‐dependent isocitrate dehydrogenases, the residues involved in dinucleotide binding and isocitrate binding are well conserved. We have developed methods for the expression in Escherichia coli and purification of the enzyme from H. volcanii . This expression was carried out in E. coli as inclusion bodies using the cytoplasmic expression vector pET3a. The enzyme was refolded by solubilisation in 8 M urea followed by dilution into a buffer containing EDTA, MgCl 2 and 3 M NaCl. Maximal activity was obtained after several hours incubation at room temperature.