A combined cell-free transcription-translation system from Saccharomyces cerevisiae for rapid and robust protein synthe.

Cell-free protein synthesis (CFPS) provides a valuable platform for understanding, using, and expanding the capabilities of the translation apparatus. For example, high-throughput CFPS is helping to address the increasing discrepancy between genome sequence data and their translation products. Here, we report the development of a combined cell-free transcription-translation (Tx/Tl) system from Saccharomyces cerevisiae that is suitable for such efforts. First, we show the ability to enable translation initiation in a cap-independent manner. The performance of various genetic elements was assessed, including 5'-UTR, 3'-UTR, and length of poly(A) tail. A specific vector harboring the 5'-UTR fragment of the Ω sequence from the tobacco mosaic virus and a poly(A) tail of 50 nucleotides led to optimal performance. Second, we developed a simple, two-step polymerase chain reaction (PCR) method for high-throughput production of linear templates for yeast CFPS. This procedure allows all functional elements needed for Tx/Tl to be added to an open-reading frame directly by overlap extension PCR. Our two-step PCR method was successfully applied to three reporter proteins: luciferase, green fluorescence protein, and chloramphenicol acetyl transferase, yielding 7 to 12.5 μg mL-1 active protein after 1.5-h batch reactions. Surprisingly, the linear templates outperformed plasmid DNA by up to 60%. Hence, the presented CFPS method has the potential to rapidly prepare tens to thousands of DNA templates without time-consuming cloning work. Further, it holds promise for fast and convenient optimization of expression constructs, study of internal ribosome entry site, and production of protein libraries for genome-scale studies. See accompanying commentary by Russ and Dueber DOI: 10.1002/biot.201400071.

[1]  A hysteretic cycle in glucose 6-phosphate metabolism observed in a cell-free yeast extract. , 1990, European journal of biochemistry.

[2]  M. Jewett,et al.  Substrate replenishment and byproduct removal improve yeast cell-free protein synthesis. , 2014, Biotechnology journal.

[3]  S. Thompson,et al.  In vivo functional analysis of the Dicistroviridae intergenic region internal ribosome entry sites , 2011, Nucleic acids research.

[4]  Hong Wang,et al.  An optimized yeast cell-free system: sufficient for translation of human papillomavirus 58 L1 mRNA and assembly of virus-like particles. , 2008, Journal of bioscience and bioengineering.

[5]  Michael C Jewett,et al.  An integrated cell-free metabolic platform for protein production and synthetic biology , 2008, Molecular systems biology.

[6]  S. Kubick,et al.  IRES-Mediated Translation of Membrane Proteins and Glycoproteins in Eukaryotic Cell-Free Systems , 2013, PloS one.

[7]  W. Miller,et al.  Cap-independent translation of plant viral RNAs. , 2006, Virus research.

[8]  G M Edelman,et al.  Transcript leader regions of two Saccharomyces cerevisiae mRNAs contain internal ribosome entry sites that function in living cells. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[9]  T. Wandless,et al.  Dicistronic regulation of fluorescent proteins in the budding yeast Saccharomyces cerevisiae , 2009, Yeast.

[10]  P. Sarnow,et al.  Translation-competent extracts from Saccharomyces cerevisiae: effects of L-A RNA, 5' cap, and 3' poly(A) tail on translational efficiency of mRNAs. , 1997, Methods.

[11]  M. Jewett,et al.  Optimized extract preparation methods and reaction conditions for improved yeast cell‐free protein synthesis , 2013, Biotechnology and bioengineering.

[12]  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 .

[13]  O. Nishimura,et al.  Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system. , 2010, Journal of biotechnology.

[14]  Michael C Jewett,et al.  Cell-free biology: exploiting the interface between synthetic biology and synthetic chemistry. , 2012, Current opinion in biotechnology.

[15]  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.

[16]  S. Thompson,et al.  Translation initiation factors are not required for Dicistroviridae IRES function in vivo. , 2009, RNA.

[17]  Kyoung-Rok Kim,et al.  Molecular characterization of an aldo-keto reductase from Marivirga tractuosa that converts retinal to retinol. , 2014, Journal of biotechnology.

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

[19]  B. Semler,et al.  Bridging IRES elements in mRNAs to the eukaryotic translation apparatus. , 2009, Biochimica et biophysica acta.

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

[21]  Y. Dorokhov,et al.  Polypurine (A)-rich sequences promote cross-kingdom conservation of internal ribosome entry , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Esther Vázquez,et al.  Microbial factories for recombinant pharmaceuticals , 2009 .

[23]  J. Swartz,et al.  Total amino acid stabilization during cell-free protein synthesis reactions. , 2006, Journal of biotechnology.

[24]  L. Maquat,et al.  Regulation of cytoplasmic mRNA decay , 2012, Nature Reviews Genetics.

[25]  T. Yamane,et al.  A Trimmed Viral Cap‐Independent Translation Enhancing Sequence for Rapid in Vitro Gene Expression , 2000, Biotechnology progress.

[26]  Amy C. Kelly,et al.  Saccharomyces cerevisiae , 2013, Prion.

[27]  L. Maquat,et al.  Regulation of cytoplasmic mRNA decay , 2012, Nature Reviews Genetics.

[28]  Kirill Alexandrov,et al.  Species-independent translational leaders facilitate cell-free expression , 2009, Nature Biotechnology.

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

[30]  S. Kubick,et al.  Cell‐free protein expression based on extracts from CHO cells , 2014, Biotechnology and bioengineering.

[31]  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.

[32]  P Sarnow,et al.  Cap-dependent and cap-independent translation by internal initiation of mRNAs in cell extracts prepared from Saccharomyces cerevisiae , 1994, Molecular and cellular biology.

[33]  Paul S. Freemont,et al.  Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology , 2013, Nucleic acids research.

[34]  O. Nishimura,et al.  Performance of expression vector, pTD1, in insect cell-free translation system. , 2006, Journal of bioscience and bioengineering.

[35]  H. Ruis,et al.  Preparation of a mRNA-dependent cell-free translation system from whole cells of Saccharomyces cerevisiae. , 1982, European journal of biochemistry.

[36]  Cem Albayrak,et al.  Cell-free co-production of an orthogonal transfer RNA activates efficient site-specific non-natural amino acid incorporation , 2013, Nucleic acids research.

[37]  Vincent Noireaux,et al.  Synthesis of 2.3 mg/ml of protein with an all Escherichia coli cell-free transcription-translation system. , 2014, Biochimie.

[38]  D. Gallie,et al.  The 5'-leader of tobacco mosaic virus promotes translation through enhanced recruitment of eIF4F. , 2002, Nucleic acids research.

[39]  E. Wahle,et al.  Control of poly(A) tail length , 2011, Wiley interdisciplinary reviews. RNA.

[40]  M. Kozak.  Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes , 1986, Cell.

[41]  Vincent Noireaux,et al.  Linear DNA for rapid prototyping of synthetic biological circuits in an Escherichia coli based TX-TL cell-free system. , 2014, ACS synthetic biology.

[42]  Kazuyuki Takai,et al.  Practical cell-free protein synthesis system using purified wheat embryos , 2010, Nature Protocols.

[43]  F. Hartl,et al.  De novo folding of GFP fusion proteins: high efficiency in eukaryotes but not in bacteria. , 2005, Journal of molecular biology.

[44]  J. Masson,et al.  Localization of a promoter in the putative internal ribosome entry site of the Saccharomyces cerevisiae TIF4631 gene. , 2004, RNA.

[45]  T. Sawasaki,et al.  Construction of an efficient expression vector for coupled transcription/translation in a wheat germ cell-free system. , 2000, Nucleic acids symposium series.

[46]  T. Hunt,et al.  Plasmid cDNA-directed protein synthesis in a coupled eukaryotic in vitro transcription-translation system. , 1992, Nucleic acids research.

[47]  Shigemichi Nishikawa,et al.  Establishment and characterization of cell-free translation/glycosylation in insect cell (Spodoptera frugiperda 21) extract prepared with high pressure treatment , 2001, Applied Microbiology and Biotechnology.

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

[49]  J. Atabekov,et al.  A tobamovirus genome that contains an internal ribosome entry site functional in vitro. , 1997, Virology.

[50]  Kim A Woodrow,et al.  Cell-free protein synthesis with prokaryotic combined transcription-translation. , 2004, Methods in molecular biology.