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| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
1
|
| pubmed:dateCreated |
1996-2-22
|
| pubmed:databankReference | |
| pubmed:abstractText |
Glutaredoxin (Grx) (12 kDa) is a hydrogen donor for ribonucleotide reductase and also a general GSH-disulfide reductase of importance for redox regulation. To overexpress human glutaredoxin in Escherichia coli, a cDNA encoding human Grx was modified and cloned into the vector pET-3d and expressed in E. coli BL21 (DE3) by IPTG induction. High-level expression of Grx was verified by GSH-disulfide oxidoreductase activity, SDS-PAGE and immunoblotting analysis. The recombinant human Grx in its reduced form was purified to homogenity with 50% yield and exhibited the same dehydroascorbate reductase and hydrogen donor activity for ribonucleotide reductase (Km approximately 0.2 microM) as the human placenta protein. Human Grx contains a total of 5 half-cystine residues including a non-conserved Cys7 residue and is easily oxidized to form dimers during storage. A Grx mutant Cys7 to Ser was generated by site-directed mutagenesis and the protein was purified to homogeneity. The mutant protein showed full activity and exhibited a much reduced tendency to form dimers compared with the wild type protein. Peptide sequencing confirmed the mutation and removal of the N-terminal Met residue in both wild type and mutant proteins. Fluorescence spectra demonstrated only tyrosine fluorescence in human Grx with a peak at 310 nm which increased 20% upon reduction and decreased by addition of GSSG demonstrating that glutathione-containing disulfides are excellent substrates.
|
| pubmed:language |
eng
|
| pubmed:journal | |
| pubmed:citationSubset |
IM
|
| pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/Cysteine,
http://linkedlifedata.com/resource/pubmed/chemical/GLRX protein, human,
http://linkedlifedata.com/resource/pubmed/chemical/Glutaredoxins,
http://linkedlifedata.com/resource/pubmed/chemical/Macromolecular Substances,
http://linkedlifedata.com/resource/pubmed/chemical/Oxidoreductases,
http://linkedlifedata.com/resource/pubmed/chemical/Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Recombinant Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Serine
|
| pubmed:status |
MEDLINE
|
| pubmed:month |
Jan
|
| pubmed:issn |
0014-5793
|
| pubmed:author | |
| pubmed:issnType |
Print
|
| pubmed:day |
2
|
| pubmed:volume |
378
|
| pubmed:owner |
NLM
|
| pubmed:authorsComplete |
Y
|
| pubmed:pagination |
69-73
|
| pubmed:dateRevised |
2007-11-15
|
| pubmed:meshHeading |
pubmed-meshheading:8549805-Amino Acid Sequence,
pubmed-meshheading:8549805-Base Sequence,
pubmed-meshheading:8549805-Cysteine,
pubmed-meshheading:8549805-Escherichia coli,
pubmed-meshheading:8549805-Gene Expression,
pubmed-meshheading:8549805-Genetic Vectors,
pubmed-meshheading:8549805-Glutaredoxins,
pubmed-meshheading:8549805-Humans,
pubmed-meshheading:8549805-Macromolecular Substances,
pubmed-meshheading:8549805-Molecular Sequence Data,
pubmed-meshheading:8549805-Mutagenesis, Site-Directed,
pubmed-meshheading:8549805-Oxidation-Reduction,
pubmed-meshheading:8549805-Oxidoreductases,
pubmed-meshheading:8549805-Proteins,
pubmed-meshheading:8549805-Recombinant Proteins,
pubmed-meshheading:8549805-Serine,
pubmed-meshheading:8549805-Structure-Activity Relationship,
pubmed-meshheading:8549805-Transfection
|
| pubmed:year |
1996
|
| pubmed:articleTitle |
High-level expression of fully active human glutaredoxin (thioltransferase) in E. coli and characterization of Cys7 to Ser mutant protein.
|
| pubmed:affiliation |
Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
|
| pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
|