THE FORMATION OF DISELENIDE BRIDGES IN PROTEINS BY INCORPORATION OF SELENOCYSTEINE RESIDUES - BIOSYNTHESIS AND CHARACTERIZATION OF (SE)(2)-THIOREDOXIN

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%.