Expression and purification of catalytically active human PHD3 in Escherichia coli
2007 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 54, no 1, 1-10 p.Article in journal (Refereed) Published
Transcription factor HIF-1 is a key regulator in cellular adaptation to hypoxia. HIF prolyl hydroxylases (PHDs) control HIF-1 accumulation by hydroxylation dependent on molecular oxygen. Due to this regulation, PHDs have been pointed out as potential drug targets. We have purified catalytically active human PHD3 after heterologous expression in Escherichia coli. Histidine-tagged enzyme was isolated as monomer by immobilized Ni-affinity chromatography followed by gel filtration. Overexpression of bacterial chaperonins GroEL/ES at 30 °C substantially increased the yield of soluble PHD3. High concentrations of salt and reducing agent during purification prevented protein aggregation. The enzyme activity with peptide derived from HIF-1α was inhibited by Zn2+, desferrioxamine and imidazole. The hydroxylation activity was verified by mass spectrometry, and Pro567 in HIF-1α was discovered as a new site of hydroxylation.
Place, publisher, year, edition, pages
2007. Vol. 54, no 1, 1-10 p.
Chaperonine, dioxygenase, EGLN, hydroxyproline, ischemia, proline
Biochemistry and Molecular Biology
Research subject Biochemistry
IdentifiersURN: urn:nbn:se:uu:diva-10913DOI: 10.1016/j.pep.2007.02.018ISI: 000246711200001PubMedID: 17434750OAI: oai:DiVA.org:uu-10913DiVA: diva2:38681