S. Muller et al., THE FORMATION OF DISELENIDE BRIDGES IN PROTEINS BY INCORPORATION OF SELENOCYSTEINE RESIDUES - BIOSYNTHESIS AND CHARACTERIZATION OF (SE)(2)-THIOREDOXIN, Biochemistry, 33(11), 1994, pp. 3404-3412
A system was devised which allows the efficient substitution of cystei
ne residues in a protein by selenocysteine. It involves overexpression
of the respective gene with the aid of the T7 promotor/ polymerase sy
stem in a cysteine auxotrophic strain. The induction of the T7 polymer
ase formation was performed in cysteine-supplemented medium followed b
y wash-out of the cysteine and production of the desired gene product
in the presence of selenocysteine. The system was applied to substitut
e the two cysteine residues in Escherichia coil thioredoxin. Analysis
of the purified gene product by electrospray mass spectrometry and HPL
C revealed that both cysteine residues were replaced in approximately
75-80% of the protein, only one cysteine residue was substituted in ab
out 5-10%, and no substitution had taken place in 12-17% of the protei
n. The occurrence of diselenide, seleno-sulfur, and disulfide bridges
in the purified gene product was revealed by ES/MS and chemical modifi
cation studies. The diselenide bridge represents an entity in protein
structures which has hitherto not been described. The redox property o
f the selenocysteine variant of thioredoxin [(Se)(2)-thioredoxin] was
found to be substantially different from that of thioredoxin. Only the
latter could be reduced under native conditions in the presence of an
excess of beta-mercaptoethanol, The oxidized (Se)(2)-thioredoxin was
then separated from the selectively reduced and carboxymethylated prot
ein by anion-exchange chromatography. The purity of the isolated (Se)(
2)-thioredoxin was at least 92%.