Ca. Padilla et al., HIGH-LEVEL EXPRESSION OF FULLY ACTIVE HUMAN GLUTAREDOXIN (THIOLTRANSFERASE) IN ESCHERICHIA-COLI AND CHARACTERIZATION OF CYS(7) TO SER MUTANT PROTEIN, FEBS letters, 378(1), 1996, pp. 69-73
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.