(477e) Azide-Bearing Amino Acid Incorporation Into Recombinant Proteins In E. Coli

Unnatural amino acids can be incorporated into recombinant proteins in vivo through the use of engineered aminoacyl-tRNA synthetases (aaRSs). In studies conducted to screen methionyl-tRNA synthetase (MetRS) mutants that allow for the incorporation of the unnatural amino acid azidonorleucine (ANL), a MetRS variant with a leucine to glycine (L13G) mutation in its methionine binding pocket was identified. Methionine auxotrophic and prototrophic bacteria were engineered by replacing their genomic MetRS allele, metG, with the mutant synthetase gene, metG*, which encodes for the MetRS L13G variant. The resulting engineered strains were able to replace methionine with ANL at high levels. Both engineered auxotrophic and prototrophic strains produced more ANL substituted protein than the wild-type strains with plasmid-borne copies of metG* in addition to genomic copies of native metG. Despite the fact that ANL was incorporated at lower levels in the prototrophic strain, more functional recombinant protein was produced in this strain versus its auxotrophic counterpart. However, both strains were able to produce recombinant proteins with sufficient azide chemical handles in order to perform click chemistry. The incorporation of azide-bearing amino acids can be utilized for various applications using the engineered MetRS L13G.