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Sequential protection-modification method for selective sulfhydryl group derivatization in proteins having more than one cysteine



Sequential protection-modification method for selective sulfhydryl group derivatization in proteins having more than one cysteine



Protein Engineering 3(7): 625-628



The method of Smith and Hartman [J. Biol. Chem., 263, 4921-4925 (1988)] for introducing the non-natural lysine analog, S-(2-aminoethyl)cysteine, into specific sites in proteins by alkylation of a genetically introduced cysteine with 2-bromoethylamine has been generalized to be applicable to proteins containing one or more endogenous cysteines. The target cysteine residue introduced at the active site of aspartate aminotransferase is protected by bound cofactor. The enzyme is partially unfolded in low concentrations of urea, and the non-active site cysteine residues derivatized by a reversible thiol protecting reagent. The active site cysteine is then exposed and alkylated in 6 M urea. Enzyme activity is regenerated by removal of the thiol protecting groups and refolding of the protein.

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Accession: 041337321

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PMID: 2217135

DOI: 10.1093/protein/3.7.625


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