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

Författare

Natalia Fedulova

Jörg Hanrieder

Göteborgs universitet

Jonas Bergquist

Lars O Emrén

Protein Expression and Purification

1046-5928 (ISSN) 1096-0279 (eISSN)

Vol. 54 1 1-10

Ämneskategorier

Biokemi och molekylärbiologi

Annan industriell bioteknik

DOI

10.1016/j.pep.2007.02.018

PubMed

17434750