Genetically expanded cell-free protein synthesis using endogenous pyrrolysyl orthogonal translation system

Yonatan Chemla, Eden Ozer, Orr Schlesinger, Vincent Noireaux, Lital Alfonta

Research output: Contribution to journalArticlepeer-review

42 Scopus citations


Cell-free protein synthesis offers a facile and rapid method for synthesizing, monitoring, analyzing, and purifying proteins from a DNA template. At the same time, genetic code expansion methods are gaining attention due to their ability to site-specifically incorporate unnatural amino acids (UAAs) into proteins via ribosomal translation. These systems are based on the exogenous addition of an orthogonal translation system (OTS), comprising an orthogonal tRNA, and orthogonal aminoacyl tRNA synthetase (aaRS), to the cell-free reaction mixture. However, these components are unstable and their preparation is labor-intensive, hence introducing a major challenge to the system. Here, we report on an approach that significantly reduces the complexity, effort and time needed to express UAA-containing proteins while increasing stability and realizing maximal suppression efficiency. We demonstrate an endogenously introduced orthogonal pair that enables the use of the valuable yet insoluble pyrrolysyl-tRNA synthetase in a cell-free system, thereby expanding the genetic repertoire that can be utilized in vitro and enabling new possibilities for bioengineering. With the high stability and efficiency of our system, we offer an improved and accessible platform for UAA incorporation into proteins.

Original languageEnglish
Pages (from-to)1663-1672
Number of pages10
JournalBiotechnology and Bioengineering
Issue number8
StatePublished - 1 Aug 2015


  • Amber suppression
  • Cell-free protein synthesis
  • Genetic code expansion
  • Pyrrolysyl-tRNA synthetase

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology


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