Simplified methodology for a modular and genetically expanded protein synthesis in cell-free systems

Yonatan Chemla, Eden Ozer, Michael Shaferman, Ben Zaad, Rambabu Dandela, Lital Alfonta

Research output: Contribution to journalArticlepeer-review


Genetic code expansion, which enables the site-specific incorporation of unnatural amino acids into proteins, has emerged as a new and powerful tool for protein engineering. Currently, it is mainly utilized inside living cells for a myriad of applications. However, the utilization of this technology in a cell-free, reconstituted platform has several advantages over living systems. The typical limitations to the employment of these systems are the laborious and complex nature of its preparation and utilization. Herein, we describe a simplified method for the preparation of this system from Escherichia coli cells, which is specifically adapted for the expression of the components needed for cell-free genetic code expansion. Besides, we propose and demonstrate a modular approach to its utilization. By this approach, it is possible to prepare and store different extracts, harboring various translational components, and mix and match them as needed for more than four years retaining its high efficiency. We demonstrate this with the simultaneous incorporation of two different unnatural amino acids into a reporter protein. Finally, we demonstrate the advantage of cell-free systems over living cells for the incorporation of δ-thio-boc-lysine into ubiquitin by using the methanosarcina mazei wild-type pyrrolysyl tRNACUA and tRNA-synthetase pair, which could not be achieved in a living cell.

Original languageAmerican English
Pages (from-to)189-196
Number of pages8
JournalSynthetic and Systems Biotechnology
Issue number4
StatePublished - 1 Dec 2019


  • Cell free system
  • Genetic code expansion
  • Simplified extract preparation
  • Thio-lysine

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Biomedical Engineering
  • Applied Microbiology and Biotechnology
  • Genetics


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