A single genomic copy of an engineered methionyl-tRNA synthetase enables robust incorporation of azidonorleucine into recombinant proteins in E. coli.
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.
Abdeljabbar
Diya M
DM
Department of Chemical Engineering, Princeton University, A207 Engineering Quadrangle, Princeton, New Jersey 08544, USA.
Klein
Thomas J
TJ
Zhang
Siyan
S
Link
A James
AJ
D004926:Escherichia coli / Q000235:genetics*; D023281:Genomics; D008718:Methionine-tRNA Ligase / Q000235:genetics* / Q000378:metabolism; D009646:Norleucine / Q000378:metabolism*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
D011994:Recombinant Proteins; D009646:Norleucine; D008718:Methionine-tRNA Ligase