Cell-Free Synthesis of Selenoproteins in High Yield and Purity for Selective Protein Tagging
| Autor: | Iresha D. Herath, Yi Jiun Tan, Daniella Goldfarb, Thomas Huber, Adarshi P. Welegedara, Angeliki Giannoulis, Mithun C. Mahawaththa, Gottfried Otting, Ansis Maleckis, Ruchira Bandara |
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| Rok vydání: | 2020 |
| Předmět: |
Selenocysteine
biology Molecular Structure 010405 organic chemistry Chemistry Stereochemistry Organic Chemistry Selenol Alkylation 010402 general chemistry Dipicolinic acid 01 natural sciences Biochemistry Maltose-Binding Proteins 0104 chemical sciences chemistry.chemical_compound Maltose-binding protein Yield (chemistry) Protein biosynthesis biology.protein Molecular Medicine Picolinic Acids Selenoproteins Molecular Biology Cysteine |
| Zdroj: | Chembiochem : a European journal of chemical biology. 22(8) |
| ISSN: | 1439-7633 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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