Abstract
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).
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Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1671–1677, July, 2015.
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Hushpulian, D.M., Zakharyants, A.A., Smirnova, N.A. et al. Reactivation of HIF prolyl hydroxylase 2 from E.coli inclusion bodies. Russ Chem Bull 64, 1671–1677 (2015). https://doi.org/10.1007/s11172-015-1058-4
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DOI: https://doi.org/10.1007/s11172-015-1058-4