Simultaneous and site-directed incorporation of an ester linkage and an azide group into a polypeptide by in vitro translation.
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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ABSTRACT: The presence of a nonproteinogenic moiety in a nonstandard peptide often improves the biological properties of the peptide. Non-standard peptide libraries are therefore used to obtain valuable molecules for biological, therapeutic, and diagnostic applications. Highly diverse non-standard peptide libraries can be generated by chemically or enzymatically modifying standard peptide libraries synthesized by the ribosomal machinery, using posttranslational modifications. Alternatively, strategies for encoding non-proteinogenic amino acids into the genetic code have been developed for the direct ribosomal synthesis of non-standard peptide libraries. In the strategies for genetic code expansion, non-proteinogenic amino acids are assigned to the nonsense codons or 4-base codons in order to add these amino acids to the universal genetic code. In contrast, in the strategies for genetic code reprogramming, some proteinogenic amino acids are erased from the genetic code and non-proteinogenic amino acids are reassigned to the blank codons. Here, we discuss the generation of genetically encoded non-standard peptide libraries using these strategies and also review recent applications of these libraries to the selection of functional non-standard peptides.Journal of nucleic acids 01/2012; 2012:713510.