Expression and purification of catalytically active human PHD3 in Escherichia coli.
Artikel i vetenskaplig tidskrift, 2007
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 degrees 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-1alpha was inhibited by Zn(2+), desferrioxamine and imidazole. The hydroxylation activity was verified by mass spectrometry, and Pro567 in HIF-1alpha was discovered as a new site of hydroxylation.
Temperature
chemistry
Chromatography
Imidazoles
Zinc
Escherichia coli
chemistry
alpha Subunit
biosynthesis
biosynthesis
Affinity
genetics
pharmacology
Hypoxia-Inducible Factor 1
Hydroxylation
chemistry
Gel
Nickel
isolation & purification
Chaperonins
Catalysis
Heat-Shock Proteins
pharmacology
biosynthesis
pharmacology
biosynthesis
Escherichia coli Proteins
chemistry
Dioxygenases
genetics
genetics
Recombinant Fusion Proteins
Hypoxia-Inducible Factor-Proline Dioxygenases
Deferoxamine
Chromatography
drug effects
Isopropyl Thiogalactoside
biosynthesis
Mass Spectrometry
isolation & purification
genetics
Amino Acid Sequence
Humans
pharmacology
genetics