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 dithiothreitol; 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 the N terminus of the P. furiosus protein were determined by Edman degradation, and its gene was cloned in Escherichia coli. The amino acid sequence derived from the DNA sequence contains the CPYC sequence, which is typical of the active site of glutaredoxin (also called thioltransferase). The C-terminal portion of the P. furiosus protein, containing 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 activity) with an optimum pH of 8.0. The expression of the P. furiosus protein, 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 filtration chromatography. The features of the P. furiosus protein here described are discussed in light of the current knowledge about the ubiquitous family of protein disulfide oxidoreductases.
Guagliardi
A
A
Dipartimento di Chimica Organica e Biologica, Università di Napoli, Italy.
de Pascale
D
D
Cannio
R
R
Nobile
V
V
Bartolucci
S
S
Rossi
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001105:Archaea / Q000378:metabolism*; D001483:Base Sequence; D002417:Cattle; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015870:Gene Expression; D005798:Genes, Bacterial; D054477:Glutaredoxins; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010088:Oxidoreductases; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013552:Swine
D004269:DNA, Bacterial; D054477:Glutaredoxins; D007328:Insulin; D009838:Oligodeoxyribonucleotides; D011506:Proteins; D011994:Recombinant Proteins; D010088:Oxidoreductases; D004229:Dithiothreitol