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Expression and puri...
Expression and purification of catalytically active human PHD3 in Escherichia coli.
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- Fedulova, Natalia, 1980- (författare)
- Uppsala universitet,Institutionen för biokemi och organisk kemi
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- Hanrieder, Jörg, 1980 (författare)
- Uppsala universitet,Gothenburg University,Göteborgs universitet,Institutionen för neurovetenskap och fysiologi, sektionen för psykiatri och neurokemi,Institute of Neuroscience and Physiology, Department of Psychiatry and Neurochemistry,University of Gothenburg,Analytisk kemi
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- Bergquist, Jonas (författare)
- Uppsala universitet,Analytisk kemi
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- Emrén, Lars O (författare)
- Uppsala universitet,Institutionen för biokemi och organisk kemi
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(creator_code:org_t)
- Elsevier BV, 2007
- 2007
- Engelska.
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Ingår i: Protein expression and purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 54:1, s. 1-10
- Relaterad länk:
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http://dx.doi.org/10...
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https://gup.ub.gu.se...
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https://doi.org/10.1...
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https://research.cha...
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https://urn.kb.se/re...
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Abstract
Ämnesord
Stäng
- 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.
Ämnesord
- NATURVETENSKAP -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)
- TEKNIK OCH TEKNOLOGIER -- Industriell bioteknik -- Annan industriell bioteknik (hsv//swe)
- ENGINEERING AND TECHNOLOGY -- Industrial Biotechnology -- Other Industrial Biotechnology (hsv//eng)
Nyckelord
- Amino Acid Sequence
- Catalysis
- Chaperonins
- biosynthesis
- genetics
- Chromatography
- Affinity
- Chromatography
- Gel
- Deferoxamine
- pharmacology
- Dioxygenases
- biosynthesis
- genetics
- isolation & purification
- Escherichia coli
- chemistry
- drug effects
- genetics
- Escherichia coli Proteins
- biosynthesis
- genetics
- Heat-Shock Proteins
- biosynthesis
- genetics
- Humans
- Hydroxylation
- Hypoxia-Inducible Factor 1
- alpha Subunit
- chemistry
- Hypoxia-Inducible Factor-Proline Dioxygenases
- Imidazoles
- pharmacology
- Isopropyl Thiogalactoside
- pharmacology
- Mass Spectrometry
- Nickel
- chemistry
- Recombinant Fusion Proteins
- biosynthesis
- chemistry
- isolation & purification
- Temperature
- Zinc
- pharmacology
- Temperature
- Biochemistry
- Biokemi
Publikations- och innehållstyp
- ref (ämneskategori)
- art (ämneskategori)
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