Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation

TLDR

Introducing chemically unique groups into proteins via non‑natural amino acids enables diverse engineering and functional studies, and incorporating azide‑functionalized amino acids in vivo allows selective labeling under native conditions. We show that azidohomoalanine is incorporated in place of methionine by E.

Abstract

The introduction of chemically unique groups into proteins by means of non-natural amino acids has numerous applications in protein engineering and functional studies. One method to achieve this involves the utilization of a non-natural amino acid by the cell's native translational apparatus. Here we demonstrate that a methionine surrogate, azidohomoalanine, is activated by the methionyl-tRNA synthetase of Escherichia coli and replaces methionine in proteins expressed in methionine-depleted bacterial cultures. We further show that proteins containing azidohomoalanine can be selectively modified in the presence of other cellular proteins by means of Staudinger ligation with triarylphosphine reagents. Incorporation of azide-functionalized amino acids into proteins in vivo provides opportunities for protein modification under native conditions and selective labeling of proteins in the intracellular environment.