Non-standard amino acid incorporation into proteins using Escherichia coli cell-free protein synthesis

Seok Hoon Hong, Yong Chan Kwon, Michael C. Jewett*

*Corresponding author for this work

Research output: Contribution to journalShort surveypeer-review

94 Scopus citations


Incorporating non-standard amino acids (NSAAs) into proteins enables new chemical properties, new structures, and new functions. In recent years, improvements in cell-free protein synthesis (CFPS) systems have opened the way to accurate and efficient incorporation of NSAAs into proteins. The driving force behind this development has been three-fold. First, a technical renaissance has enabled high-yielding (> 1 g/L) and long-lasting (> 10 h in batch operation) CFPS in systems derived from Escherichia coli. Second, the efficiency of orthogonal translation systems (OTSs) has improved. Third, the open nature of the CFPS platform has brought about an unprecedented level of control and freedom of design. Here, we review recent developments in CFPS platforms designed to precisely incorporate NSAAs. In the coming years, we anticipate that CFPS systems will impact efforts to elucidate structure/function relationships of proteins and to make biomaterials and sequence-defined biopolymers for medical and industrial applications.

Original languageEnglish (US)
Article number34
JournalFrontiers in Chemistry
Issue numberJUN
StatePublished - 2014


  • Cell-free protein synthesis
  • Genome engineering
  • Non-standard amino acids
  • Sequence-defined polymers
  • Synthetic biology

ASJC Scopus subject areas

  • General Chemistry


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