Efficient genetic encoding of phosphoserine and its nonhydrolyzable analog

Daniel T. Rogerson, Amit Sachdeva, Kaihang Wang, Tamanna Haq, Agne Kazlauskaite, Susan M. Hancock, Nicolas Huguenin-Dezot, Miratul M. K. Muqit, Andrew M. Fry, Richard Bayliss, Jason W. Chin (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

99 Citations (Scopus)

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/tRNA<inf>CUA</inf> 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.

Original languageEnglish
Pages (from-to)496-503
Number of pages8
JournalNature Chemical Biology
Volume11
Issue number7
DOIs
Publication statusPublished - Jul 2015

Fingerprint Dive into the research topics of 'Efficient genetic encoding of phosphoserine and its nonhydrolyzable analog'. Together they form a unique fingerprint.

  • No photo of Miratul Muqit

    Muqit, Miratul

    • MRC PPU - Professor (Clinical) & Personal Chair of Experimental Neurology (Clinical)

    Person: Academic

Cite this