HIGH-LEVEL EXPRESSION OF FULLY ACTIVE HUMAN GLUTAREDOXIN (THIOLTRANSFERASE) IN ESCHERICHIA-COLI AND CHARACTERIZATION OF CYS(7) TO SER MUTANT PROTEIN

Glutaredoxin (Grx) (12 kDa) is a hydrogen donor for ribonucleotide red uctase and also a general GSH-disulfide reductase of importance for re dox 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 e xpression of Grx was verified by GSH-disulfide oxidoreductase activity , SDS-PAGE and immunoblotting analysis. The recombinant human Grx in i ts reduced form was purified to homogenity with 50% yield and exhibite d the same dehydroascorbate reductase and hydrogen donor activity for ribonucleotide reductase (K-m similar to 0.2 mu M) as the human placen ta protein. Human Grx contains a total of 5 half-cystine residues incl uding a non-conserved Cys(7) residue and is easily oxidized to form di mers during storage. A Grx mutant Cys(7) 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 tende ncy to form dimers compared with the wild type protein. Peptide sequen cing confirmed the mutation and removal of the N-terminal Met residue in both wild type and mutant proteins. Fluorescence spectra demonstrat ed only tyrosine fluorescence in human Grx with a peak at 310 nm which increased 20% upon reduction and decreased by addition of GSSG demons trating that glutathione-containing disulfides are excellent substrate s.