title: Synthesis of 2.3 mg/ml of protein with an all Escherichia coli cell-free transcription-translation system.
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-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.
authors: School of Physics and Astronomy, University of Minnesota, 116 Church Street SE, Minneapolis 55455, Minnesota, United States.
authors: Filippo
authors: F
authors: Caschera
authors: School of Physics and Astronomy, University of Minnesota, 116 Church Street SE, Minneapolis 55455, Minnesota, United States. Electronic address: noireaux@umn.edu.
authors: Vincent
authors: V
authors: Noireaux
mesh_terms: D002458:Cell Fractionation; D004734:Energy Metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D014176:Protein Biosynthesis; D014158:Transcription, Genetic
chemical_list: D029968:Escherichia coli Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008320:Maltose
keywords: Cell-free protein synthesis; Escherichia coli extract; Long-lived cell-free transcription–translation; Maltose; Synthetic biology