Expression, Purification and Characterization of Human PHD1 in Escherichia coli

• Published in: Journal of biochemistry (Tokyo) Vol. 144; no. 5; pp. 555 - 561
• Main Authors: Li, Xian Y, Takasaki, Chikahisa, Satoh, Yuhei, Kimura, Shigenobu, Yasumoto, Ken-ichi, Sogawa, Kazuhiro
• Format: Journal Article
• Language: English
• Published: England Japanese Biochemical Society 01.11.2008; Oxford University Press
• Subjects: Amino Acid Sequence; Animals; Dioxygenases - genetics; Dioxygenases - isolation & purification; Dioxygenases - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; HIF prolyl hydroxylase; HIF-1α stabilization; Humans; Hypoxia-Inducible Factor 1, alpha Subunit - metabolism; Hypoxia-Inducible Factor-Proline Dioxygenases; Ions - metabolism; Isoenzymes - genetics; Isoenzymes - isolation & purification; Isoenzymes - metabolism; Metals - metabolism; Mice; Molecular Sequence Data; Mutation; Nuclear Proteins - genetics; Nuclear Proteins - isolation & purification; Nuclear Proteins - metabolism; Phosphorylation; Procollagen-Proline Dioxygenase; protein kinase Cα; recombinant PHD1; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; transition metal ions; protein kinase Cα; HIF prolyl hydroxylase; HIF-1α stabilization; recombinant PHD1; transition metal ions
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/jb/mvn102

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 IC₅₀ 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.