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  • Cell‐Free Synthesis of Sele...
    Welegedara, Adarshi P.; Maleckis, Ansis; Bandara, Ruchira; Mahawaththa, Mithun C.; Dilhani Herath, Iresha; Jiun Tan, Yi; Giannoulis, Angeliki; Goldfarb, Daniella; Otting, Gottfried; Huber, Thomas

    Chembiochem : a European journal of chemical biology, April 16, 2021, Letnik: 22, Številka: 8
    Journal Article

    The selenol group of selenocysteine is much more nucleophilic than the thiol group of cysteine. Selenocysteine residues in proteins thus offer reactive points for rapid post‐translational modification. Herein, we show that selenoproteins can be expressed in high yield and purity by cell‐free protein synthesis by global substitution of cysteine by selenocysteine. Complete alkylation of solvent‐exposed selenocysteine residues was achieved in 10 minutes with 4‐chloromethylene dipicolinic acid (4Cl‐MDPA) under conditions that left cysteine residues unchanged even after overnight incubation. GdIII−GdIII distances measured by double electron–electron resonance (DEER) experiments of maltose binding protein (MBP) containing two selenocysteine residues tagged with 4Cl‐MDPA‐GdIII were indistinguishable from GdIII−GdIII distances measured of MBP containing cysteine reacted with 4Br‐MDPA tags. 34Se‐lective protein tagging: Cell‐free protein synthesis is used to produce selenoproteins in high yield and purity. Selenocysteine has long been recognized as offering an exquisitely reactive chemical handle for chemical modifications of proteins, and we show that the alkylation of solvent‐exposed selenol groups with a chloro‐alkyl group proceeds quickly under conditions that leaves cysteine residues unchanged.