Journal
NATURE CHEMICAL BIOLOGY
Volume 11, Issue 7, Pages 496-+Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/NCHEMBIO.1823
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Funding
- UK Medical Research Council [U105181009, UD99999908]
- European Research Council
- Wellcome Trust [101022/Z/13/Z]
- Worldwide Cancer Research grant [13-0042]
- Cancer Research UK Programme Award [C24461/A12772]
- Wellcome Trust [101022/Z/13/Z] Funding Source: Wellcome Trust
- Cancer Research UK [12772] Funding Source: researchfish
- Medical Research Council [MC_U105181009, MC_UP_A024_1008] Funding Source: researchfish
- Parkinson's UK [H-1403, G-1506] Funding Source: researchfish
- Worldwide Cancer Research [13-0042] Funding Source: researchfish
- MRC [MC_U105181009, MC_UP_A024_1008] Funding Source: UKRI
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Serine phosphorylation is a key post-translational modification that regulates diverse biological processes. Powerful analytical methods have identified thousands of phosphorylation sites, but many of their functions remain to be deciphered. A key to understanding the function of protein phosphorylation is access to phosphorylated proteins, but this is often challenging or impossible. Here we evolve an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair that directs the efficient incorporation of phosphoserine (pSer (1)) into recombinant proteins in Escherichia coli. Moreover, combining the orthogonal pair with a metabolically engineered Escherichia coli enables the site-specific incorporation of a nonhydrolyzable analog of pSer. Our approach enables quantitative decoding of the amber stop codon as pSer, and we purify, with yields of several milligrams per liter of culture, proteins bearing biologically relevant phosphorylations that were previously challenging or impossible to access-including phosphorylated ubiquitin and the kinase Nek7, which is synthetically activated by a genetically encoded phosphorylation in its activation loop.
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