Cell-Free Synthesis of Selenoproteins in High Yield and Purity for Selective Protein Tagging.
Chembiochem
; 22(8): 1480-1486, 2021 04 16.
Article
em En
| MEDLINE
| ID: mdl-33319405
ABSTRACT
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.
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Base de dados:
MEDLINE
Assunto principal:
Ácidos Picolínicos
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Selenoproteínas
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Proteínas Ligantes de Maltose
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article