Padilla C A, Spyrou G, Holmgren A
Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
FEBS Lett. 1996 Jan 2;378(1):69-73. doi: 10.1016/0014-5793(95)01413-6.
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.
谷氧还蛋白（Grx）（12 kDa）是核糖核苷酸还原酶的氢供体，也是对氧化还原调节很重要的一种通用谷胱甘肽二硫化物还原酶。为了在大肠杆菌中过表达人谷氧还蛋白，编码人Grx的cDNA被修饰并克隆到载体pET-3d中，通过IPTG诱导在大肠杆菌BL21（DE3）中表达。通过谷胱甘肽二硫化物氧化还原酶活性、SDS-PAGE和免疫印迹分析验证了Grx的高水平表达。还原形式的重组人Grx以50%的产率纯化至均一，并且对核糖核苷酸还原酶（Km约为0.2 microM）表现出与人胎盘蛋白相同的脱氢抗坏血酸还原酶和氢供体活性。人Grx总共含有5个半胱氨酸残基，包括一个非保守的Cys7残基，并且在储存过程中很容易被氧化形成二聚体。通过定点诱变产生了一个Cys7突变为Ser的Grx突变体，并将该蛋白纯化至均一。与野生型蛋白相比，突变蛋白显示出完全活性，并且形成二聚体的倾向大大降低。肽测序证实了野生型和突变型蛋白中N端Met残基的突变和去除。荧光光谱显示人Grx中只有酪氨酸荧光，峰值在310 nm，还原时增加20%，加入GSSG后降低，表明含谷胱甘肽的二硫化物是优良的底物。
