Expression and purification of catalytically active human PHD3 in Escherichia coli.
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.
Fedulova
Natalia
N
Department of Biochemistry and Organic Chemistry, Uppsala University, Box 576, SE-751 23 Uppsala, Sweden.
Hanrieder
Jörg
J
Bergquist
Jonas
J
Emrén
Lars O
LO
D000595:Amino Acid Sequence; D002384:Catalysis; D018833:Chaperonins / Q000096:biosynthesis / Q000235:genetics; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003676:Deferoxamine / Q000494:pharmacology; D049308:Dioxygenases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D006900:Hydroxylation; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D007093:Imidazoles / Q000494:pharmacology; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D013058:Mass Spectrometry; D009532:Nickel / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013696:Temperature; D015032:Zinc / Q000494:pharmacology
D029968:Escherichia coli Proteins; C028715:GroE protein, E coli; D006360:Heat-Shock Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007093:Imidazoles; D011993:Recombinant Fusion Proteins; D007544:Isopropyl Thiogalactoside; C029899:imidazole; D009532:Nickel; D049308:Dioxygenases; C488894:EGLN3 protein, human; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D018833:Chaperonins; D003676:Deferoxamine; D015032:Zinc