Expression, purification and characterization of human PHD1 in Escherichia coli

Xian Y. Li, Chikahisa Takasaki, Yuhei Satoh, Shigenobu Kimura, Ken Ichi Yasumoto, Kazuhiro Sogawa

Research output: Contribution to journalArticlepeer-review

7 Citations (Scopus)


The hypoxia-inducible factors (HIFs) play a central role in oxygen homeostasis. HIF prolyl hydroxylases (PHDs) modify HIFα subunits and thereby target them for proteasomal degradation. Mammalian PHDs comprise three isozymes, PHD1, PHD2 and PHD3, and belong to the iron(II)-2-oxoglutarate- dependent dioxygenase family. We have expressed full-length human PHD1 in Escherichia coli, and purified it to apparent homogeneity by immobilized Ni-affinity chromatography, cation-exchange HPLC followed by gel filtration. Fe2+ was found to have EC50 value of 0.64 μM and the purified enzyme showed maximal activity at 10 μM Fe2+. The IC 50 values for transition metal ions, Co2+, Ni2+ and Cu2+, were 58, 35 and 220 μM, respectively, in the presence of 100 μM Fe2+. Mn2+ did not affect the activity <1 mM. Many transcription-related proteins are regulated by phosphorylation. Thus, recombinant PHD1 was examined for in vitro phosphorylation using protein kinase A, protein kinase Cα, casein kinase I and II and Erk2. The protein was most strongly phosphorylated by protein kinase Cα, and the phosphorylation sites were found to be Ser-132, Ser-226 and Ser-234. Mutation of Ser-132 or Ser-234 to Asp or Glu diminished the enzymatic activity to 25-60%, while mutation of Ser-226 scarcely influenced the activity.

Original languageEnglish
Pages (from-to)555-561
Number of pages7
JournalJournal of biochemistry
Issue number5
Publication statusPublished - 2008 Nov


  • HIF prolyl hydroxylase
  • HIF-1α stabilization
  • Protein kinase Cα
  • Recombinant PHD1
  • Transition metal ions

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


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