NADP-dependent isocitrate dehydrogenase from the halophilic archaeon Haloferax volcanii: cloning, sequence determination and overexpression in Escherichia coli.
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 lambda EMBL3 library. Analysis of the nucleotide sequence revealed an open reading frame of 1260 bp encoding a protein of 419 amino acids with 45837 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.
Camacho
Mónica
M
División de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Alicante, Ap. 99, E-03080, Spain.
Rodríguez-Arnedo
Adoración
A
Bonete
María José
MJ
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda; D003001:Cloning, Molecular; D017124:Conserved Sequence; D030561:Databases, Nucleic Acid; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D019622:Haloferax volcanii / Q000201:enzymology / Q000235:genetics*; D007521:Isocitrate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+)