title: Efficient genetic encoding of phosphoserine and its nonhydrolyzable analog.
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.
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK.
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Rogerson
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK.
authors.forename: Amit
authors.initials: A
authors.lastname: Sachdeva
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK.
authors.forename: Kaihang
authors.initials: K
authors.lastname: Wang
authors.affiliation: Department of Biochemistry, University of Leicester, Leicester, UK.
authors.forename: Tamanna
authors.initials: T
authors.lastname: Haq
authors.affiliation: Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK.
authors.forename: Agne
authors.initials: A
authors.lastname: Kazlauskaite
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Hancock
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Huguenin-Dezot
authors.affiliation: 1] Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK. [2] College of Medicine, Dentistry and Nursing, University of Dundee, Dundee, UK.
authors.forename: Miratul M K
authors.initials: MM
authors.lastname: Muqit
authors.affiliation: Department of Biochemistry, University of Leicester, Leicester, UK.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Fry
authors.affiliation: Department of Biochemistry, University of Leicester, Leicester, UK.
authors.forename: Richard
authors.initials: R
authors.lastname: Bayliss
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK.
authors.forename: Jason W
authors.initials: JW
authors.lastname: Chin
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D018388:Codon, Terminator / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005815:Genetic Code; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000072379:NIMA-Related Kinases; D009690:Nucleic Acid Conformation; D010766:Phosphorylation; D010768:Phosphoserine / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D018388:Codon, Terminator; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D025801:Ubiquitin; D010768:Phosphoserine; C512685:NEK7 protein, human; D000072379:NIMA-Related Kinases; D017346:Protein Serine-Threonine Kinases; D000604:Amino Acyl-tRNA Synthetases