Synthesis of 2.3 mg/ml of protein with an all Escherichia coli cell-free transcription-translation system

Filippo Caschera, Vincent Noireaux

Research output: Contribution to journalArticlepeer-review

180 Scopus citations


Cell-free protein synthesis is becoming a useful technique for synthetic biology. As more applications are developed, the demand for novel and more powerful in vitro expression systems is increasing. In this work, an all Escherichia coli cell-free system, that uses the endogenous transcription and translation molecular machineries, is optimized to synthesize up to 2.3 mg/ml of a reporter protein in batch mode reactions. A new metabolism based on maltose allows recycling of inorganic phosphate through its incorporation into newly available glucose molecules, which are processed through the glycolytic pathway to produce more ATP. As a result, the ATP regeneration is more efficient and cell-free protein synthesis lasts up to 10 h. Using a commercial E. coli strain, we show for the first time that more than 2 mg/ml of protein can be synthesized in run-off cell-free transcription-translation reactions by optimizing the energy regeneration and waste products recycling. This work suggests that endogenous enzymes present in the cytoplasmic extract can be used to implement new metabolic pathways for increasing protein yields. This system is the new basis of a cell-free gene expression platform used to construct and to characterize complex biochemical processes in vitro such as gene circuits.

Original languageEnglish (US)
Pages (from-to)162-168
Number of pages7
Issue number1
StatePublished - Apr 2014

Bibliographical note

Funding Information:
We thank Paul Jardine and Shelley Grimes for use of the cell press and technical help. We thank Jonathan Garamella, Ryan Marshall, Mark Rustad and Charles Godfrey for technical help. This research was supported by the Defense Advanced Research Projects Agency (DARPA/MTO) Living Foundries program, contract number HR0011-12-C-0065 (DARPA/CMO) and by the Office of Naval Research award number N00014-13-1-0074. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing officially policies, either expressly or implied, of the Defense Advanced Research Projects Agency or the U.S. Government.


  • Cell-free protein synthesis
  • Escherichia coli extract
  • Long-lived cell-free transcription-translation
  • Maltose
  • Synthetic biology


Dive into the research topics of 'Synthesis of 2.3 mg/ml of protein with an all Escherichia coli cell-free transcription-translation system'. Together they form a unique fingerprint.

Cite this