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

Significance Statement

 

Cell-free expression technology is an emerging field with many potential applications. Such systems are design to synthesize protein in vitro from synthetic DNA programs. All the molecular machineries used to express the protein are extracted from a living organism, such as E. coli. In this work we present an all E. coli cell-free transcription-translation system fueled by an improved metabolic pathway designed to recycle inorganic phosphate, the byproduct of the transcription-translation processes. The core innovation is the increased level of ATP available for the protein synthesis during cell-free expression. The system is improved by addition of maltose, a disaccharide that is hydrolyzed by cytoplasmic endogenous enzymes that simultaneously attached the inorganic phosphate and generate metabolic intermediates processed in the glycolytic pathway to sustain the production of ATP.

So far this system is the most effective in term of ATP regeneration. The yield of cell-free protein synthesis in batch mode reactions is the largest observed so far (2.3 mg/ml). We envision many potential applications for our system, from proteomic studies to synthetic biology toward metabolic engineering for instance.

In conclusion, we have designed a powerful E. coli cell-free expression system, based on a novel metabolic pathway. Overall, we have increased knowledge on the dynamics of cell-free expression systems and we aim to use this platform for studies in synthetic biology and build-up complex systems from the bottom-up in the laboratory.

Synthesis of 2.3 l of protein with an all Escherichia coli cell-free transcription-translation system

Journal Reference

Caschera F, Noireaux V.

Biochimie. 2014 Apr;99:162-8.

School of Physics and Astronomy, University of Minnesota, 116 Church Street SE, Minneapolis 55455, Minnesota, United States. Electronic address: [email protected]

 

Abstract

 

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-translationreactions 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.

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