Efficient genetic encoding of phosphoserine and its nonhydrolyzable analog
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.
© 2015 Macmillan Publishers Limited. Received 14 February 2015; accepted 15 April 2015; published online 1 June 2015. We are grateful to the UK Medical Research Council Laboratory of Molecular Biology (MRC-LMB) Mass Spectrometry for extensive assistance. M. Richards (University of Leicester) for the Nek7 plasmid, T. Elliott (MRC-LMB), J. Madrzak (MRC-LMB) and M. Mahesh (MRC-LMB) for assistance. This work was supported by grants to J.W.C. from the UK Medical Research Council (U105181009 and UD99999908) and the European Research Council. M.M.K.M. is supported by the Wellcome Trust (101022/Z/13/Z), J. Macdonald Menzies Charitable Trust and Tenovus (Scotland). A.F.M. is supported by a Worldwide Cancer Research grant (13-0042) and R.B. by a Cancer Research UK Programme Award (C24461/A12772). Author Contributions: D.T.R. and J.W.C. conceived the experimental strategy, analyzed the data and wrote the paper with input from other authors. D.T.R. performed all the selections, system characterization and most phosphoprotein expressions and purifications. S.M.H. and D.T.R. characterized the starting system. K.W., A.S., D.T.R. and N.H.-D. developed and characterized the expression system. T.H. and D.T.R. performed and analyzed the Nek7 experiments with guidance from A.M.F. and R.B. A.K. performed and analyzed the ubiquitin assays with guidance from M.M.K.M. The authors declare no competing financial interests.
Accepted Version - emss-63029.pdf
Supplemental Material - nchembio.1823-S1.pdf