Journal
CELL CHEMICAL BIOLOGY
Volume 25, Issue 9, Pages 1067-+Publisher
CELL PRESS
DOI: 10.1016/j.chembiol.2018.05.013
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Funding
- Medical Research Council, UK [MC_U105181009, MC_UP_A024_1008]
- ERC Advanced Grant (SGCR) [669351]
- MRC case studentship (Nikon)
- Konrad-Adenauer Foundation
- EMBO Fellowship [ALTF 297-2015]
- NSF [1523390]
- MRC [MC_UP_A024_1008, MC_U105181009] Funding Source: UKRI
- Div Of Biological Infrastructure
- Direct For Biological Sciences [1523390] Funding Source: National Science Foundation
- European Research Council (ERC) [669351] Funding Source: European Research Council (ERC)
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Protein phosphorylation regulates diverse processes in eukaryotic cells. Strategies for installing site-specific phosphorylation in target proteins in eukaryotic cells, through routes that are orthogonal to enzymatic post-translational modification, would provide a powerful route for defining the consequences of particular phosphorylations. Here we show that the SepRS(v1.0)/tRNA(CUA)(v1.0) pair (created from the Methanococcus maripaludis phosphoseryl-transfer RNA synthetase [MmSepRS]/Methanococcus janaschii [Mj]tRNA(GCA)(Cys) pair) is orthogonal in mammalian cells. We create a eukaryotic elongation factor 1 alpha (EF-1 alpha) variant, EF-1 alpha-Sep, that enhances phosphoserine incorporation, and combine this with a mutant of eRF1, and manipulations of the cell's phosphoserine biosynthetic pathway, to enable the genetically encoded incorporation of phosphoserine and its non-hydrolyzable phosphonate analog. Using this approach we demonstrate synthetic activation of a protein kinase in mammalian cells.
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