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 cysteine residues in a protein by selenocysteine. It involves overexpression of the respective gene with the aid of the T7 promotor/polymerase system in a cysteine auxotrophic strain. The induction of the T7 polymerase formation was performed in cysteine-supplemented medium followed by wash-out of the cysteine and production of the desired gene product in the presence of selenocysteine. The system was applied to substitute the two cysteine residues in Escherichia coli thioredoxin. Analysis of the purified gene product by electrospray mass spectrometry and HPLC revealed that both cysteine residues were replaced in approximately 75-80% of the protein, only one cysteine residue was substituted in about 5-10%, and no substitution had taken place in 12-17% of the protein. The occurrence of diselenide, seleno-sulfur, and disulfide bridges in the purified gene product was revealed by ES/MS and chemical modification studies. The diselenide bridge represents an entity in protein structures which has hitherto not been described. The redox property of 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 protein by anion-exchange chromatography. The purity of the isolated (Se)2-thioredoxin was at least 92%.
Müller
S
S
Lehrstuhl für Mikrobiologie, Universität München, Germany.
Senn
H
H
Gsell
B
B
Vetter
W
W
Baron
C
C
Böck
A
A
D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry*; D007525:Isoelectric Focusing; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D012350:RNA, Transfer, Cys / Q000378:metabolism; D011994:Recombinant Proteins; D012643:Selenium / Q000737:chemistry*; D017279:Selenocysteine / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013879:Thioredoxins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D014764:Viral Proteins
D012350:RNA, Transfer, Cys; D011994:Recombinant Proteins; D014764:Viral Proteins; D017279:Selenocysteine; D013879:Thioredoxins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D012643:Selenium; D003545:Cysteine