Simultaneous and site-directed incorporation of an ester linkage and an azidegroup into a polypeptide by in vitrotranslation

Abstract

A method is presented by which an azide-containing side chain can be introduced into any internal position of a polypeptide chain by in vitro translation. For this, 2′-deoxy-cytidylyl-(3′→5′)-adenosine was acylated on the 3′(2′)-hydroxyl group of adenosine with 6-azido-2(S)-hydroxyhexanoic acid (AHHA), an α-hydroxy- and ε-azide derivative of L-lysine. The acylated dinucleotide was enzymatically ligated with a tRNA transcript to provide chemically stable E. coli suppressor AHHA-tRNA^Cys(CUA). The esterase 2 gene from Alicyclobacillus acidocaldarius was modified by the amber stop codon (UAG) on position 118. Using AHHA-tRNA^Cys(CUA) in an E. coli in vitro translation/transcription system, the site-directed introduction of an azide group linked to a backbone ester into the esterase polypeptide was achieved. The yield of the synthesized modified protein reached 80% compared to translation of the native esterase. Subsequently, azide coupling with an alkyne-modified oligodeoxynucleotide demonstrated the feasibility of this approach for conjugation of polypeptides.