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Cell‐Free Synthesis of Selenoproteins in High Yield and Purity for Selective Protein Tagging
Author(s) -
Welegedara Adarshi P.,
Maleckis Ansis,
Bandara Ruchira,
Mahawaththa Mithun C.,
Dilhani Herath Iresha,
Jiun Tan Yi,
Giannoulis Angeliki,
Goldfarb Daniella,
Otting Gottfried,
Huber Thomas
Publication year - 2021
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.202000785
Subject(s) - selenocysteine , cysteine , chemistry , maltose binding protein , biochemistry , stereochemistry , recombinant dna , enzyme , gene , fusion protein
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. Gd III −Gd III distances measured by double electron–electron resonance (DEER) experiments of maltose binding protein (MBP) containing two selenocysteine residues tagged with 4Cl‐MDPA‐Gd III were indistinguishable from Gd III −Gd III distances measured of MBP containing cysteine reacted with 4Br‐MDPA tags.