期刊
CHEMBIOCHEM
卷 22, 期 8, 页码 1480-1486出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.202000785
关键词
electron paramagnetic resonance spectroscopy; paramagnetic tags; protein labeling; selenocysteine; selenoproteins
资金
- Australian Research Council [DP200100348, DP210100088, FL170100019, CE200100012]
- Australian Research Council [FL170100019, DP200100348] Funding Source: Australian Research Council
The study demonstrates that selenoproteins can be efficiently expressed by replacing cysteine with selenocysteine, and rapid alkylation of exposed selenocysteine residues can be achieved under 4Cl-MDPA conditions without affecting cysteine residues.
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.
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