WE HAVE DEVELOPED A BACTERIAL EXPRESSION SYSTEM WHICH ALLOWS THE EFFICIENT SUBSTITUTION OF CYSTEINE RESIDUES IN A PROTEIN BY SELENOCYSTEINE (SEE: MILLER, S., SENN, H., GSELL, B., VETTER, W. AND BVCK, A. (1994), BIOCHEMISTRY 33, 3404-3412). IT INVOLVES OVEREXPRESSION OF THE RESPECTIVE GENE WITH THE AID OF THE T7 PROMOTER/POLYMERIZE SYSTEM IN A CYSTEINE AUXOTROPH STRAIN. THE SYSTEM WAS APPLIED TO SUBSTITUTE THE TWO CYSTEINE RESIDUES IN E.COLI THIOREDOXIN. (SE)2-THIOREDOXIN WAS ISOLATED AND BIOCHEMICALLY CHARACTERIZED. USING THIS METHOD, L-[3-77SE]CYSTEINE PROVIDIE BY SIR WILL BE INCORPORATED INTO THIOREDOXIN WHICH WILL REPLACE THE DISULFIDE BRIDGE BY A 77SE-ENRICHED DISELENIDE BRIDGE. THE ISOLATED (77SE)2-THIOREDOXIN WILL BE STUDIED BY 77SE NMR SPECTROSCOPY. THE DISULFIDE IN THIOREDOXIN SERVES AS A TWO ELECTRON REDOX CARRIER FOR THE REDUCTION OF NUCLEOTIDES TO DEOXY NUCLEOTIDES. WE WILL DEVELOP A METHOD DIRECTLY DETERMINE THE REDOX STATUS OF DISULFIDES IN PROTEINS USING 77SE NMR SPECTROSCOPY.
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