title: Expanding the genetic code of Escherichia coli with phosphoserine.
abstract: O-Phosphoserine (Sep), the most abundant phosphoamino acid in the eukaryotic phosphoproteome, is not encoded in the genetic code, but synthesized posttranslationally. Here, we present an engineered system for specific cotranslational Sep incorporation (directed by UAG) into any desired position in a protein by an Escherichia coli strain that harbors a Sep-accepting transfer RNA (tRNA(Sep)), its cognate Sep-tRNA synthetase (SepRS), and an engineered EF-Tu (EF-Sep). Expanding the genetic code rested on reengineering EF-Tu to relax its quality-control function and permit Sep-tRNA(Sep) binding. To test our system, we synthesized the activated form of human mitogen-activated ERK activating kinase 1 (MEK1) with either one or two Sep residues cotranslationally inserted in their canonical positions (Sep(218), Sep(222)). This system has general utility in protein engineering, molecular biology, and disease research.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
authors.forename: Hee-Sung
authors.initials: HS
authors.lastname: Park
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Hohn
authors.forename: Takuya
authors.initials: T
authors.lastname: Umehara
authors.forename: Li-Tao
authors.initials: LT
authors.lastname: Guo
authors.forename: Edith M
authors.initials: EM
authors.lastname: Osborne
authors.forename: Jack
authors.initials: J
authors.lastname: Benner
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Noren
authors.forename: Jesse
authors.initials: J
authors.lastname: Rinehart
authors.forename: Dieter
authors.initials: D
authors.lastname: Söll
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D000926:Anticodon; D002701:Chloramphenicol / Q000494:pharmacology; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D018388:Codon, Terminator; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D005815:Genetic Code; D005818:Genetic Engineering; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D010444:Peptide Elongation Factor Tu; D010768:Phosphoserine / Q000378:metabolism*; D015202:Protein Engineering; D046188:Protein Modification, Translational; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012345:RNA, Transfer, Amino Acid-Specific / Q000235:genetics / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012350:RNA, Transfer, Cys / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D046249:Transfer RNA Aminoacylation
chemical_list: D000926:Anticodon; D018388:Codon, Terminator; D012329:RNA, Bacterial; D012345:RNA, Transfer, Amino Acid-Specific; D012346:RNA, Transfer, Amino Acyl; D012350:RNA, Transfer, Cys; D011993:Recombinant Fusion Proteins; D010768:Phosphoserine; D002701:Chloramphenicol; D015500:Chloramphenicol O-Acetyltransferase; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D010444:Peptide Elongation Factor Tu; D000604:Amino Acyl-tRNA Synthetases