High‐level expression of fully active human glutaredoxin (thioltransferase) in E. coli and characterization of Cys7 to Ser mutant protein

FEBS Letters

Spyrou, Giannis¹  Alicia Padilla, C.¹  Holragren, Arne¹ 

[1] Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-17177 Stockholm, Sweden

关键词: Glutaredoxin;    Thioltransferase;    Thiol-disulfide;    Recombinant protein;    Site-directed mutagenesis;    Fluorescence;    BSA;    bovine serum albumin;    DTT;    dithiolthreitol;    GSH;    glutathione;    GSSG;    oxidized glutathione;    HED;    β-ydroxyethylene disulfide;    PMSF;    phenylmethylsulfonyl fluoride;    DHA;    dehydro l-ascorbate;    IPTG;    isopropyl;    β-d-thiogalactopyranoside;    NMR;    nuclear magnetic resonance;    TGF;    transforming growth factor;   

DOI  :  10.1016/0014-5793(95)01413-6

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 (K _m ∼ 0.2 μM) 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 glutathionecontaining disulfides are excellent substrates.

【 授权许可】

Unknown