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 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.

[1]  Dong-Myung Kim,et al.  Prolonged production of proteins in a cell-free protein synthesis system using polymeric carbohydrates as an energy source , 2011 .

[2]  G. Zubay,et al.  In vitro synthesis of protein in microbial systems. , 1973, Annual review of genetics.

[3]  V. Noireaux,et al.  An E. coli cell-free expression toolbox: application to synthetic gene circuits and artificial cells. , 2012, ACS synthetic biology.

[4]  Andrew Buchanan,et al.  Coping with complexity: Machine learning optimization of cell‐free protein synthesis , 2011, Biotechnology and Bioengineering.

[5]  Michael C Jewett,et al.  Molecular Systems Biology Peer Review Process File in Vitro Integration of Ribosomal Rna Synthesis, Ribosome Assembly, and Translation Transaction Report , 2022 .

[6]  J. Swartz,et al.  Energizing cell-free protein synthesis with glucose metabolism. , 2005, Biotechnology and bioengineering.

[7]  Su-Jin Oh,et al.  Cell-free platforms for flexible expression and screening of enzymes. , 2013, Biotechnology advances.

[8]  Yutetsu Kuruma,et al.  A synthetic biology approach to the construction of membrane proteins in semi-synthetic minimal cells. , 2009, Biochimica et biophysica acta.

[9]  Steen Rasmussen,et al.  Protocells : bridging nonliving and living matter , 2008 .

[10]  Vincent Noireaux,et al.  Study of messenger RNA inactivation and protein degradation in an Escherichia coli cell-free expression system , 2010, Journal of biological engineering.

[11]  Vincent Noireaux,et al.  A vesicle bioreactor as a step toward an artificial cell assembly. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[12]  Y.‐H.P. Zhang,et al.  Simpler Is Better: High-Yield and Potential Low-Cost Biofuels Production through Cell-Free Synthetic Pathway Biotransformation (SyPaB) , 2011 .

[13]  Vincent Noireaux,et al.  Assembly of MreB filaments on liposome membranes: a synthetic biology approach. , 2012, ACS synthetic biology.

[14]  M. Isalan,et al.  Engineering Gene Networks to Emulate Drosophila Embryonic Pattern Formation , 2005, PLoS biology.

[15]  R. Bar-Ziv,et al.  Protein nanomachines assembly modes: cell-free expression and biochip perspectives. , 2013, Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology.

[16]  Richard M. Murray,et al.  Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology , 2013, Journal of visualized experiments : JoVE.

[17]  Michael L Simpson,et al.  Cell-free synthetic biology: a bottom-up approach to discovery by design , 2006, Molecular systems biology.

[18]  David K. Karig,et al.  Expression optimization and synthetic gene networks in cell-free systems , 2011, Nucleic acids research.

[19]  T. Katayama,et al.  Cooperative working of bacterial chromosome replication proteins generated by a reconstituted protein expression system , 2013, Nucleic acids research.

[20]  Dan S. Tawfik,et al.  Man-made cell-like compartments for molecular evolution , 1998, Nature Biotechnology.

[21]  Ardemis A. Boghossian,et al.  Label-free, single protein detection on a near-infrared fluorescent single-walled carbon nanotube/protein microarray fabricated by cell-free synthesis. , 2011, Nano letters.

[22]  Kate Thomas.  The Past , 2015 .

[23]  C. J. Murray,et al.  Microscale to Manufacturing Scale-up of Cell-Free Cytokine Production—A New Approach for Shortening Protein Production Development Timelines , 2011, Biotechnology and bioengineering.

[24]  Pasquale Stano,et al.  Approaches to semi-synthetic minimal cells: a review , 2005, Naturwissenschaften.

[25]  Talmon Arad,et al.  Cell-free co-synthesis of protein nanoassemblies: tubes, rings, and doughnuts. , 2007, Nano letters.

[26]  John L Markley,et al.  Cell-free protein production and labeling protocol for NMR-based structural proteomics , 2004, Nature Methods.

[27]  R. Bar-Ziv,et al.  Cell-free protein synthesis and assembly on a biochip. , 2012, Nature nanotechnology.

[28]  A. Spirin.  High-throughput cell-free systems for synthesis of functionally active proteins. , 2004, Trends in biotechnology.

[29]  Dong-Myung Kim,et al.  Methods for energizing cell-free protein synthesis. , 2009, Journal of bioscience and bioengineering.

[30]  R. Bar-Ziv,et al.  Principles of cell-free genetic circuit assembly , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[31]  Cheemeng Tan,et al.  Molecular crowding shapes gene expression in synthetic cellular nanosystems , 2013, Nature nanotechnology.

[32]  Y.‐H.P. Zhang,et al.  Cell-free protein synthesis energized by slowly-metabolized maltodextrin , 2009, BMC biotechnology.

[33]  A. Di Maio,et al.  Di Maio, A , 2012 .

[34]  Takeshi Omasa,et al.  Synthetic metabolic engineering-a novel, simple technology for designing a chimeric metabolic pathway , 2012, Microbial Cell Factories.

[35]  Tomio Ogasawara,et al.  A cell-free protein synthesis system for high-throughput proteomics , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[36]  Thomas E. Landrain,et al.  A new frontier in synthetic biology: automated design of small RNA devices in bacteria. , 2013, Trends in genetics : TIG.

[37]  Tetsuya Yomo,et al.  In vitro evolution of α-hemolysin using a liposome display , 2013, Proceedings of the National Academy of Sciences.

[38]  Rui Gan,et al.  Cell-free protein synthesis: applications come of age. , 2012, Biotechnology advances.

[39]  Mitchel J Doktycz,et al.  Thrombin-mediated transcriptional regulation using DNA aptamers in DNA-based cell-free protein synthesis. , 2014, ACS synthetic biology.

[40]  Sven Panke,et al.  In silico assessment of cell‐free systems , 2012, Biotechnology and bioengineering.

[41]  Jim Berg,et al.  A genetically encoded fluorescent reporter of ATP/ADP ratio , 2008, Nature Methods.

[42]  Vincent Noireaux,et al.  Genome replication, synthesis, and assembly of the bacteriophage T7 in a single cell-free reaction. , 2012, ACS synthetic biology.

[43]  Michele Forlin,et al.  Fluorescent proteins and in vitro genetic organization for cell-free synthetic biology. , 2013, ACS synthetic biology.

[44]  Jim Swartz,et al.  Developing cell-free biology for industrial applications , 2006, Journal of Industrial Microbiology and Biotechnology.

[45]  Kazufumi Hosoda,et al.  Replication of Genetic Information with Self‐Encoded Replicase in Liposomes , 2008, ChemBioChem.

[46]  Geoffrey Chang,et al.  The past, present and future of cell-free protein synthesis. , 2005, Trends in biotechnology.

[47]  Dong-Myung Kim,et al.  Prolonging cell-free protein synthesis with a novel ATP regeneration system. , 1999, Biotechnology and bioengineering.

[48]  Vincent Noireaux,et al.  Development of an artificial cell, from self-organization to computation and self-reproduction , 2011 .

[49]  Bradley C. Bundy,et al.  Escherichia coli‐based cell‐free synthesis of virus‐like particles , 2008, Biotechnology and bioengineering.

[50]  Dong-Myung Kim,et al.  Regeneration of adenosine triphosphate from glycolytic intermediates for cell-free protein synthesis. , 2001, Biotechnology and bioengineering.

[51]  M. Hulko,et al.  Cell-free expression of a mammalian olfactory receptor and unidirectional insertion into small unilamellar vesicles (SUVs). , 2013, Biochimie.

[52]  Vincent Noireaux,et al.  Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70 , 2010, Journal of biological engineering.

[53]  Henrike Niederholtmeyer,et al.  Implementation of cell-free biological networks at steady state , 2013, Proceedings of the National Academy of Sciences.

[54]  T. Shimazu,et al.  Assay of inorganic phosphate in the mild pH range, suitable for measurement of glycogen phosphorylase activity. , 1985, Analytical biochemistry.

[55]  D. Baigl,et al.  Modification-free photocontrol of β-lactam conversion with spatiotemporal resolution. , 2012, ACS synthetic biology.

[56]  Jung-Won Keum,et al.  Prolonged cell‐free protein synthesis using dual energy sources: Combined use of creatine phosphate and glucose for the efficient supply of ATP and retarded accumulation of phosphate , 2007, Biotechnology and bioengineering.