An Economical Method for Cell‐Free Protein Synthesis using Glucose and Nucleoside Monophosphates

Cell‐free protein synthesis reactions have not been seriously considered as a viable method for commercial protein production mainly because of high reagent costs and a lack of scalable technologies. Here we address the first issue by presenting a cell‐free protein synthesis system with comparable protein yields that removes the most expensive substrates and lowers the cell‐free reagent cost by over 75% (excluding extract, polymerase, and plasmid) while maintaining high energy levels. This system uses glucose as the energy source and nucleoside monophosphates (NMPs) in place of nucleoside triphosphates (NTPs) as the nucleotide source. High levels of nucleoside triphosphates are generated from the monophosphates within 20 min, and the subsequent energy charge is similar in reactions beginning with either NTPs or NMPs. Furthermore, significant levels (>0.2 mM) of all NTPs are still available at the end of a 3‐h incubation, and the total nucleotide pool is stable throughout the reaction. The glucose/NMP reaction was scaled up to milliliter scale using a thin film approach. Significant yields of active protein were observed for two proteins of vastly different size: chloramphenicol acetyl transferase (CAT, 25 kDa) and β‐galactosidase (472 kDa). The glucose/NMP cell‐free reaction system dramatically reduces reagent costs while supplying high protein yields.

[1]  James Swartz,et al.  Maintaining rapid growth in moderate-density Escherichia coli fermentations. , 2005, Biotechnology and bioengineering.

[2]  J. Swartz,et al.  High-Density, Defined Media Culture for the Production ofEscherichia coliCell Extracts , 2003 .

[3]  M. Jewett,et al.  Substrate replenishment extends protein synthesis with an in vitro translation system designed to mimic the cytoplasm , 2004, Biotechnology and bioengineering.

[4]  Yasuo Shinohara,et al.  Cell‐free protein synthesis on a microchip , 2002, Proteomics.

[5]  D. E. Atkinson.  The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. , 1968, Biochemistry.

[6]  R G Kim,et al.  Expression-independent consumption of substrates in cell-free expression system from Escherichia coli. , 2000, Journal of biotechnology.

[7]  James Swartz,et al.  Amino acid stabilization for cell-free protein synthesis by modification of the Escherichia coli genome. , 2004, Metabolic engineering.

[8]  E. Henshaw,et al.  Nucleoside diphosphate regulation of overall rates of protein biosynthesis acting at the level of initiation. , 1985, The Journal of biological chemistry.

[9]  Junhao Yang,et al.  Rapid expression of vaccine proteins for B-cell lymphoma in a cell-free system. , 2005, Biotechnology and bioengineering.

[10]  T. Yamane,et al.  Phosphatase-immunodepleted cell-free protein synthesis system. , 1998, Journal of biotechnology.

[11]  Dong-Myung Kim,et al.  Prolonging Cell‐Free Protein Synthesis by Selective Reagent Additions , 2000, Biotechnology progress.

[12]  Alex Eon-duval,et al.  Purification of pharmaceutical-grade plasmid DNA by anion-exchange chromatography in an RNase-free process. , 2004, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[13]  W. V. Shaw.  Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. , 1975, Methods in enzymology.

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

[15]  Dominic Esposito,et al.  A novel cell-free protein synthesis system. , 2004, Journal of biotechnology.

[16]  M. Jewett,et al.  Mimicking the Escherichia coli cytoplasmic environment activates long‐lived and efficient cell‐free protein synthesis , 2004, Biotechnology and bioengineering.

[17]  Teruyuki Nagamune,et al.  Protein productivity of cell-free translation was improved by removing phosphatase from wheat germ extract with immunoprecipitation , 1998 .

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

[19]  R. Lakshmi,et al.  Extraction of superior-quality plasmid DNA by a combination of modified alkaline lysis and silica matrix. , 1999, Analytical biochemistry.

[20]  Dong-Myung Kim,et al.  Oxalate improves protein synthesis by enhancing ATP supply in a cell-free system derived from Escherichia coli , 2000, Biotechnology Letters.

[21]  J. Swartz,et al.  Streamlining Escherichia Coli S30 Extract Preparation for Economical Cell‐Free Protein Synthesis , 2008, Biotechnology progress.

[22]  C. Anderson,et al.  Preparation of a cell-free protein-synthesizing system from wheat germ. , 1983, Methods in enzymology.

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

[24]  T. Kigawa,et al.  Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis , 1995, Journal of biomolecular NMR.

[25]  M. Jewett,et al.  Rapid Expression and Purification of 100 nmol Quantities of Active Protein Using Cell‐Free Protein Synthesis , 2008, Biotechnology progress.

[26]  A. Spirin,et al.  A continuous cell-free translation system capable of producing polypeptides in high yield. , 1988, Science.

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

[28]  J. Swartz,et al.  Enhancing multiple disulfide bonded protein folding in a cell‐free system , 2004, Biotechnology and bioengineering.

[29]  Daisuke Kiga,et al.  An engineered Escherichia coli tyrosyl–tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germ cell-free system , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[30]  Dong-Myung Kim,et al.  Efficient production of a bioactive, multiple disulfide‐bonded protein using modified extracts of Escherichia coli , 2004, Biotechnology and bioengineering.