IMP-6609

IDH1

Recombinant IDH1 (R100A) protein was expressed in E. coli cells as the full length protein (accession number AAH93020.1) with a point mutation Arg100Ala and a C-terminal 6xHis Tag. The molecular weight of the protein is 47.7 kDa.

IDH1 (Isocitrate Dehydrogenase (NADP(+)) 1, Cytosolic, also known as HEL-216, HEL-S-26, IDCD, IDH, IDP, IDPC, PICD) is a member of isocitrate dehydrogenases, which catalyze the oxidative decarboxylation of isocitrate to 2-oxoglutarate. IDH1 is the NADP(+)-dependent isocitrate dehydrogenase found in the cytoplasm and peroxisomes. It contains the PTS-1 peroxisomal targeting signal sequence. The presence of this enzyme in peroxisomes suggests roles in the regeneration of NADPH for intraperoxisomal reductions, such as the conversion of 2, 4-dienoyl-CoAs to 3-enoyl-CoAs, as well as in peroxisomal reactions that consume 2-oxoglutarate, namely the alpha-hydroxylation of phytanic acid. The cytoplasmic enzyme serves a significant role in cytoplasmic NADPH production. Mutations in human cytosolic isocitrate dehydrogenase I (IDH1) occur somatically in >70% of grade II-III gliomas and secondary glioblastomas, and in 8.5% of acute myeloid leukemias (AML). Mutations have also been reported in cancers of the colon and prostate. To date, mutations in at least four active site arginine residues IDH1 R100, IDH1 R132, IDH2 R140, and IDH2 R172 have been shown to result in the neomorphic production of R(-)-2-hydroxyglutarate (2HG), although these mutants lack the wild-type enzyme's ability to convert isocitrate to a-ketoglutarate (a-KG, 2OG). Among of them, IDH1 R100A is affected in adult glioma.

E. coli

Human

His

Recombinant IDH1 (R100A) protein is supplied at a concentration of 1.2 μg/μL in 25 mM Tris pH 8.0, 300 mM NaCl, 5% glycerol.

47.7 kDa

>90%

Enzyme kinetics, screening inhibitors, and selectivity profiling

Recombinant proteins in solution are temperature sensitive and must be stored at -80 centigrade to prevent degradation. Avoid repeated freeze/thaw cycles and keep on ice when not in storage.

Recombinant IDH1 (R100A) protein gel. 12.5% SDS-PAGE Coomassie staining. MW: 47.7 kDa Purity: >90%

Recombinant IDH1 (R100A) protein activity assay. 10 μM NADPH and 1 μM a-KG were incubated with 100 nM IDH1 (R100A) protein in 200 μL reaction system containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10 mM MgCl2 and 0.03% BSA (room temperature). Depletion of NADPH was monitored continuously at Abs 340 nm for 20 min.