Design of Orthogonal Pairs for Protein Translation: Selection Systems for Genetically Encoding Noncanonical Amino Acids in E. coli

  • Jelena Jaric
  • Nediljko BudisaEmail author
Part of the Springer Protocols Handbooks book series (SPH)


The expansion of the genetic code is gradually becoming a core discipline in synthetic biology. Residue-specific incorporation of noncanonical amino acids (ncAAs) into proteins allows facile alteration and enhancement of protein properties. There are two distinct in vivo approaches available for their cotranslational incorporation. For isostructural noncanonical amino acids, residue-specific replacement of canonical amino acids is performed with the supplementation-based incorporation method (SPI) using auxotrophic host strains. On the other hand, orthogonal ncAAs are incorporated into the proteins site specifically in response to stop or quadruplet codons (stop codon suppression (SCS)) using orthogonal aminoacyl-tRNA synthetase/tRNA pairs (o-pair). Frequently used o-pair is based on the tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii (MjTyrRS). To evolve a new orthogonal aminoacyl-tRNA synthetase (aaRS), which recognizes exclusively the noncanonical amino acid, the most straightforward solution is to produce a library of MjTyrRS mutants, containing randomized residues in the amino acid-binding site, on the basis of available crystal structure. The library is transformed into Escherichia coli and three rounds of positive and negative selection are performed in order to select for desired MjTyrRS variant which uniquely charges the tRNA with the ncAA of interest. Here, we provide a protocol with detailed description how to perform positive and negative selection with chloramphenicol acetyltransferase and barnase, respectively.


Amber suppressor Methanocaldococcus jannaschii tRNATyrCUAopt Methanocaldococcus jannaschii tyrosyl-tRNA synthetase Noncanonical amino acid Orthogonal pair Positive and negative selection Stop codon suppression approach 


  1. 1.
    Budisa N (2013) Expanded genetic code for the engineering of ribosomally synthetized and post-translationally modified peptide natural products (RiPPs). Curr Opin Biotechnol 24:591–598CrossRefPubMedGoogle Scholar
  2. 2.
    Hoesl MG, Budisa N (2012) Recent advances in genetic code engineering in Escherichia coli. Curr Opin Biotechnol 23:751–757CrossRefPubMedGoogle Scholar
  3. 3.
    Link AJ, Mock ML, Tirrell DA (2003) Non-canonical amino acids in protein engineering. Curr Opin Biotechnol 14:603–609CrossRefPubMedGoogle Scholar
  4. 4.
    Liu CC, Schultz PG (2010) Adding new chemistries to the genetic code. Annu Rev Biochem 79:413–444CrossRefPubMedGoogle Scholar
  5. 5.
    Wang L, Brock A, Herberich B, Schultz PG (2001) Expanding the genetic code of Escherichia coli. Science 292:498–500CrossRefPubMedGoogle Scholar
  6. 6.
    Zhang Y, Wang L, Schultz PG, Wilson IA (2005) Crystal structures of apo wild-type M. jannaschii tyrosyl-tRNA synthetase (TyrRS) and an engineered TyrRS specific for O-methyl-L-tyrosine. Protein Sci 14:1340–1349CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Guo J, Melancon CE III, Lee HS, Groff D, Schultz PG (2009) Evolution of amber suppressor tRNAs for efficient bacterial production of proteins containing nonnatural amino acids. Angew Chem Int Ed 48:9148–9151CrossRefGoogle Scholar

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© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  1. 1.Technische Universität Berlin, Fakultät II, Institut für Chemie, Sekretariat L 1BerlinGermany

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