Abstract:
: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 E. 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.
journal_name
Nat Chem Bioljournal_title
Nature chemical biologyauthors
Rogerson DT,Sachdeva A,Wang K,Haq T,Kazlauskaite A,Hancock SM,Huguenin-Dezot N,Muqit MM,Fry AM,Bayliss R,Chin JWdoi
10.1038/nchembio.1823subject
Has Abstractpub_date
2015-07-01 00:00:00pages
496-503issue
7eissn
1552-4450issn
1552-4469pii
nchembio.1823journal_volume
11pub_type
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