Residue-specific incorporation of noncanonical amino acids into model proteins using an Escherichia coli cell-free transcription-translation system

Emanuel G. Worst, Matthias P. Exner, Alessandro de Simone, Marc Schenkelberger, Vincent Noireaux, Nediljko Budisa, Albrecht Ott

Research output: Contribution to journalArticlepeer-review

17 Scopus citations


The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Original languageEnglish (US)
Article numbere54273
JournalJournal of Visualized Experiments
Issue number114
StatePublished - Jul 27 2016

Bibliographical note

Funding Information:
E.G. Worst and A. Ott acknowledge financial support by the Deutsche Forschungsgemeinschaft (DFG) within the collaborative research center SFB 1027 as well as Saarland University. E.G. Worst, A. Ott and V. Noireaux further acknowledge financial aid by the Human Frontiers Science Program Organization (HFSPO). The authors thank Tobias Baumann and Stefan Oehm (Institute of Chemistry, Technische Universität Berlin) for critical reading.

Publisher Copyright:
© 2016 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License.


  • Bioengineering
  • Cell-Free expression
  • Escherichia coli cell extract
  • L-Arginine analog
  • L-Canavanine
  • Molecular biology
  • Noncanonical amino acid analog
  • Residue-Specific incorporation
  • Synthetic biology
  • Unnatural amino acid


Dive into the research topics of 'Residue-specific incorporation of noncanonical amino acids into model proteins using an Escherichia coli cell-free transcription-translation system'. Together they form a unique fingerprint.

Cite this