Simultaneous and site-directed incorporation of an ester linkage and an azide group into a polypeptide by in vitro translation

Laboratorium für Biochemie, Universität Bayreuth, Universitätstrasse 30, 95440 Bayreuth, Germany.
Organic & Biomolecular Chemistry (Impact Factor: 3.49). 10/2009; 7(20):4218-24. DOI: 10.1039/b909188b
Source: PubMed

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 alpha-hydroxy- and epsilon-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.

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