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Journal of Biochemistry Advance Access published online on August 17, 2008
Journal of Biochemistry, doi:10.1093/jb/mvn102
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© 2008 The Japanese Biochemical Society

Expression, purification and characterization of human PHD1 in Escherichia coli

Xian Y. Lia, Chikahisa Takasakia,*, Yuhei Satoha, Shigenobu Kimurab, Ken-ichi Yasumotoa and Kazuhiro Sogawaa

a Department of Biomolecular Science, Graduate School of Life Sciences, Tohoku University, Aoba-ku Sendai 980-8578, Japan
b Department of Biomolecular Functional Engineering, Faculty of Engineering, Ibaraki University, Hitachi 316-8511, Japan

*Corresponding author: Dr. Chikahisa Takasaki. Address: Department of Biomolecular Science, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku Sendai 980-8578, Japan, Tel : +81-22-795-6592, Fax: +81-22-795-6594, e-mail address: c-takasaki{at}bio.chem.tohoku.ac.jp

Received July 18, 2008; Accepted August 6, 2008


  Abstract

The hypoxia-inducible factors (HIFs) play a central role in oxygen homeostasis. HIF prolyl hydroxylases (PHDs) modify HIF{alpha} 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 below 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 C{alpha}, casein kinase I and II and Erk2. The protein was most strongly phosphorylated by protein kinase C{alpha}, 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.

Key Words: HIF-1{alpha} stabilization, HIF prolyl hydroxylase, protein kinase C{alpha}, recombinant PHD1, transition metal ions


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