PMID:21868676
| Citation |
Park, HS, Hohn, MJ, Umehara, T, Guo, LT, Osborne, EM, Benner, J, Noren, CJ, Rinehart, J and Söll, D (2011) Expanding the genetic code of Escherichia coli with phosphoserine. Science 333:1151-4 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1126/science.1207203 |
| Keywords |
Amino Acyl-tRNA Synthetases/metabolism; Anticodon; Chloramphenicol/pharmacology; Chloramphenicol O-Acetyltransferase/genetics; Codon, Terminator; Drug Resistance, Bacterial; Escherichia coli/drug effects; Escherichia coli/genetics; Escherichia coli/metabolism; Genetic Code; Genetic Engineering; Humans; MAP Kinase Kinase 1/biosynthesis; MAP Kinase Kinase 1/chemistry; MAP Kinase Kinase 1/genetics; Peptide Elongation Factor Tu; Phosphoserine/metabolism; Protein Engineering; Protein Modification, Translational; RNA, Bacterial/genetics; RNA, Bacterial/metabolism; RNA, Transfer, Amino Acid-Specific/genetics; RNA, Transfer, Amino Acid-Specific/metabolism; RNA, Transfer, Amino Acyl/metabolism; RNA, Transfer, Cys/genetics; Recombinant Fusion Proteins/biosynthesis; Transfer RNA Aminoacylation |
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