Reactivation of HIF prolyl hydroxylase 2 from E.coli inclusion bodies
| Autor: | Irina G. Gazaryan, A.A. Zakharyants, Natalia Moroz, Andrey A. Poloznikov, Vladimir I. Tishkov, D. M. Hushpulian, N. A. Smirnova |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
chemistry.chemical_classification
Gene isoform Peptide General Chemistry Molecular biology Inclusion bodies Dithiothreitol law.invention Hydroxylation 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Enzyme chemistry Hypoxia-inducible factors Biochemistry law 030220 oncology & carcinogenesis Recombinant DNA 030217 neurology & neurosurgery |
| Zdroj: | Russian Chemical Bulletin. 64:1671-1677 |
| ISSN: | 1573-9171 1066-5285 |
| DOI: | 10.1007/s11172-015-1058-4 |
| Popis: | Hypoxia inducible factor (HIF) prolyl hydroxylases (PHD) belong to α-ketoglutarate-dependent non-heme iron dioxygenases catalyzing hydroxylation of HIF prolines. The catalytic domains of all three enzyme isoforms (PHD1–3) were expressed in E.coli with the highest expression level being observed for the PHD2 isoform. In all cases, the recombinant portion of protein was mainly expressed as insoluble inclusion bodies. PHD was reactivated by refolding from inclusion bodies. To optimize the refolding procedure, a novel continuous assay based on measurement of ferrocyanide oxidation activity in the presence of HIF peptide or protein was developed. The comparison between the purified soluble enzyme samples and the PHD2 samples reactivated from inclusion bodies showed a 4–5-fold higher specific activity of the latter (5 mol min−1 vol) in the α-ketoglutarate consumption determined by fluorescence detection of ketoglutarate—o-phenylene diamine adduct. The PHD2 isoform is highly hydrophobic, has to be handled in high-molarity buffer solutions to prevent aggregation and precipitation, and is inactivated rapidly in the absence of dithiothreitol (DTT). |
| Databáze: | OpenAIRE |
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