High-level expression of fully active human glutaredoxin (thioltransferase) in E. coli and characterization of Cys7 to Ser mutant protein.
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.
Padilla
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Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
Spyrou
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Holmgren
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003545:Cysteine; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D054477:Glutaredoxins; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine; D013329:Structure-Activity Relationship; D014162:Transfection
C516005:GLRX protein, human; D054477:Glutaredoxins; D046911:Macromolecular Substances; D011506:Proteins; D011994:Recombinant Proteins; D012694:Serine; D010088:Oxidoreductases; D003545:Cysteine