A single genomic copy of an engineered methionyl-tRNA synthetase enables robust incorporation of azidonorleucine into recombinant proteins in E. coli

Diya M. Abdeljabbar, Thomas J. Klein, Siyan Zhang, Aaron James Link

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

(Chemical Equation Presented) Engineered aminoacyl-tRNA synthetases have been used to enable the incorporation of many unnatural amino acids into recombinant proteins in vivo. In the majority of these studies, the engineered synthetase is harbored on a plasmid while the host retains a wild-type copy of the synthetase in its genome. Herein, we construct a strain carrying a single genomic copy of a methionyl-tRNA synthetase (MetRS) gene, metG*, engineered to enable the incorporation of azidonorleucine (ANL) into proteins. The resulting strain, M15MA metG*, is capable of both supporting robust cell growth and enabling the production of >20 mg/L culture of a recombinant protein, murine dihydrofolate reductase, containing ANL. The extent of replacement of methionine with ANL in this protein is 90%. Using this strain, we also produce ANL-containing OmpC, an outer membrane protein, and demonstrate that the surface of cells displaying this protein can be covalently modified using copper-catalyzed azide-alkyne cycloaddition. Since this mutant MetRS has been introduced into the genome, as opposed to a plasmid, M15MA metG* is genetically stable.

Original languageEnglish (US)
Pages (from-to)17078-17079
Number of pages2
JournalJournal of the American Chemical Society
Volume131
Issue number47
DOIs
StatePublished - Dec 2 2009

All Science Journal Classification (ASJC) codes

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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