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Asymmetric enrichment of L-azidohomoalanine incorporation into the asparaginyl residues of recombinant proteins expressed in Saccharomyces cerevisiae. The incorporation of unnatural amino acids into recombinant proteins is an attractive approach for protein engineering.

The previous methods for the incorporation of L-azidohomoalanine into the asparaginyl residues of recombinant proteins were based on in vitro chemical ligation of peptides to form the alpha-imido derivative, followed by the chemoselective enzymatic or chemical reduction to form the free imide.

Here, we describe an alternative strategy for the incorporation of the azo form of alanine, L-azidohomoalanine, into recombinant proteins. We demonstrate that the success of this approach depends on the maintenance of the Asn residue in the nascent peptide as an alpha-amino group.

This method eliminates the requirement to form the alpha-azido form of alanine, thus providing a potentially inexpensive means to incorporate L-azidohomoalanine into proteins of interest.

Moreover, we demonstrate that this method of incorporating L-azidohomoalanine into recombinant proteins can also be applied in cellular contexts.