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

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 t...

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Bibliographic Details
Published in:Biochimie 2014-04, Vol.99, p.162-168
Main Authors: Caschera, Filippo, Noireaux, Vincent
Format: Article
Language:English
Subjects:
Cell Fractionation
Cell-free protein synthesis
Energy Metabolism
Escherichia coli
Escherichia coli - enzymology
Escherichia coli - genetics
Escherichia coli extract
Escherichia coli Proteins - chemistry
Escherichia coli Proteins - metabolism
Green Fluorescent Proteins - biosynthesis
Hydrogen-Ion Concentration
Kinetics
Long-lived cell-free transcription–translation
Maltose
Maltose - metabolism
Protein Biosynthesis
Synthetic biology
Transcription, Genetic
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Summary: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. [Display omitted] •All E. coli cell-free transcription–translation system.•Enhanced in vitro protein synthesis through a novel maltose metabolism.•Inorganic phosphate efficiently recycled to sustain prolonged protein synthesis.•2.3 mg/ml of total proteins produced in batch mode reactions.•Long-lived cell-free expression, up to 10 h in batch mode.
ISSN:0300-9084
1638-6183
DOI:10.1016/j.biochi.2013.11.025