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

Org Biomol Chem. 2009 Oct 21;7(20):4218-24. doi: 10.1039/b909188b. Epub 2009 Aug 12.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acylation
  • Azides / metabolism*
  • Base Sequence
  • Binding Sites
  • Codon, Terminator / genetics
  • Codon, Terminator / metabolism
  • Escherichia coli / metabolism
  • Esterases / chemistry
  • Esterases / metabolism
  • Esters / metabolism*
  • Lysine / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Peptides / chemistry*
  • Protein Biosynthesis*
  • Protein Conformation
  • RNA, Transfer / genetics
  • RNA, Transfer / metabolism
  • Time Factors

Substances

  • Azides
  • Codon, Terminator
  • Esters
  • Peptides
  • RNA, Transfer
  • Esterases
  • Lysine