THE PURIFICATION, CLONING, AND HIGH-LEVEL EXPRESSION OF A GLUTAREDOXIN-LIKE PROTEIN FROM THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS-FURIOSUS

A protein has been purified to homogeneity from crude extracts of the hyperthermophilic archaeon Pyrococcus furiosus based on its ability to catalyze the reduction of insulin disulfides in the presence of dithi othreitol; the protein has a molecular mass of 24.8 kDa and a pi of 4. 9, and it is highly heat-stable. The first 29 amino acid residues at t he N terminus of the P. furiosus protein were determined by Edman degr adation, and its gene was cloned in Escherichia coli, The amino acid s equence derived from the DNA sequence contains the CPYC sequence, whic h is typical of the active site of glutaredoxin (also called thioltran sferase), The C-terminal portion of the P. furiosus protein, containin g the conserved sequence, shows sequence similarity with glutaredoxins from different sources. The P. furiosus protein can reduce disulfide bonds in L-cystine in the presence of GSH (the thioltransferase activi ty) with an optimum pH of 8.0, The expression of the P. furiosus prote in, with full activity, in E. coli at a very high level (21% of total soluble protein) is described; the recombinant protein was purified to homogeneity by merely two successive heat treatments and gel filtrati on chromatography. The features of the P. furiosus protein here descri bed are discussed in light of the current knowledge about the ubiquito us family of protein disulfide oxidoreductases.