Condensation of an Additive-Free Cell Extract to Mimic the Conditions of Live Cells

The cellular environment differs from that of reconstituted materials mainly because of the presence of highly condensed biomacromolecules. To mimic the environment and conditions in living cells, we developed a method to prepare additive-free, highly concentrated cell extracts. First, we verified the requirement for specific salts and buffers for functional cell-free translation extracts. The S30 fraction of Escherichia coli cell extracts without additives exhibited sufficient cell-free protein production. Next, we established a method to accumulate biological components by gradual evaporation by using a vacuum desiccator. Bovine serum albumin, green fluorescent protein, alkaline phosphatase, and a diluted reconstituted protein expression system were successfully condensed in their active forms using this method. The protein concentration of the prepared cell extract was elevated to 180 mg/mL, which was expected to contain approximately 260 mg/mL macromolecules, without the loss of cell-free protein expression activity. Such a condensed cell extract may be useful for investigating the differences between cells and reconstituted materials and may contribute to the development of methods to synthesize cells from cell extracts in the future.

[1]  Gaetano T Montelione,et al.  Cold-shock induced high-yield protein production in Escherichia coli , 2004, Nature Biotechnology.

[2]  A. Minton,et al.  How can biochemical reactions within cells differ from those in test tubes? , 2006, Journal of Cell Science.

[3]  Yasuhiko Yoshida,et al.  Cell‐free production and stable‐isotope labeling of milligram quantities of proteins , 1999, FEBS letters.

[4]  Takuya Ueda,et al.  Cell-free translation reconstituted with purified components , 2001, Nature Biotechnology.

[5]  Takuya Ueda,et al.  Global analysis of chaperone effects using a reconstituted cell-free translation system , 2012, Proceedings of the National Academy of Sciences.

[6]  Anders Pedersen,et al.  Rational improvement of cell-free protein synthesis. , 2011, New biotechnology.

[7]  T. Terwilliger,et al.  Engineering and characterization of a superfolder green fluorescent protein , 2006, Nature Biotechnology.

[8]  A. Kornberg,et al.  Replication initiated at the origin (oriC) of the E. coli chromosome reconstituted with purified enzymes , 1984, Cell.

[9]  Kenichi Yoshikawa,et al.  Gene Expression within Cell‐Sized Lipid Vesicles , 2003, Chembiochem : a European journal of chemical biology.

[10]  A. Spirin,et al.  Continuous-exchange protein-synthesizing systems. , 2007, Methods in molecular biology.

[11]  Adrian H Elcock,et al.  Models of macromolecular crowding effects and the need for quantitative comparisons with experiment. , 2010, Current opinion in structural biology.

[12]  M. Waegele,et al.  Effect of macromolecular crowding on protein folding dynamics at the secondary structure level. , 2009, Journal of molecular biology.

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

[14]  W. Zillig,et al.  Dissociation and reconstitution of active DNA‐dependent RNA‐polymerase from E. coli , 1970, FEBS letters.

[15]  T. Yamane,et al.  An increased rate of cell-free protein synthesis by condensing wheat-germ extract with ultrafiltration membranes. , 1994, Bioscience, biotechnology, and biochemistry.

[16]  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.

[17]  M Wakabayashi,et al.  Synthesis of functional protein in liposome. , 2001, Journal of bioscience and bioengineering.

[18]  Kenichi Yoshikawa,et al.  Giant Liposome as a Biochemical Reactor: Transcription of DNA and Transportation by Laser Tweezers , 2001 .

[19]  Dan Luo,et al.  Cell-Free Protein Expression under Macromolecular Crowding Conditions , 2011, PloS one.

[20]  K. Yoshikawa,et al.  Cell-Sized confinement in microspheres accelerates the reaction of gene expression , 2012, Scientific Reports.

[21]  P. Walde,et al.  Building artificial cells and protocell models: experimental approaches with lipid vesicles. , 2010, BioEssays : news and reviews in molecular, cellular and developmental biology.

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

[23]  Yasuhiko Yamamoto,et al.  Five Amino Acid Residues Responsible for the High Stability of Hydrogenobacter thermophilus Cytochrome c552 , 2005, Journal of Biological Chemistry.

[24]  S. Zimmerman,et al.  Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli. , 1991, Journal of molecular biology.

[25]  Harold P. Erickson,et al.  Reconstitution of Contractile FtsZ Rings in Liposomes , 2008, Science.

[26]  H. Taguchi,et al.  A systematic survey of in vivo obligate chaperonin‐dependent substrates , 2010, The EMBO journal.

[27]  Shigemichi Nishikawa,et al.  Direct preparation of giant proteo-liposomes by in vitro membrane protein synthesis. , 2008, Journal of biotechnology.

[28]  G. Ourisson,et al.  Towards Proto‐Cells: “Primitive” Lipid Vesicles Encapsulating Giant DNA and Its Histone Complex , 2001, Cellular & molecular biology letters.

[29]  Ikuo Morita,et al.  Direct formation of proteo-liposomes by in vitro synthesis and cellular cytosolic delivery with connexin-expressing liposomes. , 2009, Biomaterials.