A cell-free protein synthesis system for high-throughput proteomics

We report a cell-free system for the high-throughput synthesis and screening of gene products. The system, based on the eukaryotic translation apparatus of wheat seeds, has significant advantages over other commonly used cell-free expression systems. To maximize the yield and throughput of the system, we optimized the mRNA UTRs, designed an expression vector for large-scale protein production, and developed a new strategy to construct PCR-generated DNAs for high-throughput production of many proteins in parallel. The resulting system achieves high-yield expression and can maintain productive translation for 14 days. Additionally, in the integration of a PCR-directed system for template creation, at least 50 genes can be translated in parallel, yielding between 0.1 and 2.3 mg of protein by one person within 2 days. Assessment of correct protein folding by the products of this high-throughput protein-expression system were performed by enzymatic assays of kinases and by NMR spectroscopic analysis. The cell-free system, reported here, bypasses many of the time-consuming cloning steps of conventional expression systems and lends itself to a robotic automation for the high-throughput expression of proteins.

[1]  B. Paterson,et al.  Efficient translation of tobacco mosaic virus RNA and rabbit globin 9S RNA in a cell-free system from commercial wheat germ. , 1973, Proceedings of the National Academy of Sciences of the United States of America.

[2]  C. Kurland.  Translational accuracy in vitro , 1982, Cell.

[3]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

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

[5]  P G Schultz,et al.  A general method for site-specific incorporation of unnatural amino acids into proteins. , 1989, Science.

[6]  H. Sambrook.  Molecular cloning : a laboratory manual. Cold Spring Harbor, NY , 1989 .

[7]  R. Burgess,et al.  Use of in vitro protein synthesis from polymerase chain reaction-generated templates to study interaction of Escherichia coli transcription factors with core RNA polymerase and for epitope mapping of monoclonal antibodies. , 1991, The Journal of biological chemistry.

[8]  J. Sninsky,et al.  Recent advances in the polymerase chain reaction , 1991, Science.

[9]  V. Walbot,et al.  Identification of the motifs within the tobacco mosaic virus 5'-leader responsible for enhancing translation. , 1992, Nucleic acids research.

[10]  R. Wetzel,et al.  Inclusion body formation and protein stability in sequence variants of interleukin-1 beta. , 1993, The Journal of biological chemistry.

[11]  V. Gurevich.  Use of bacteriophage RNA polymerase in RNA synthesis. , 1996, Methods in enzymology.

[12]  M. Ehrenberg,et al.  Rate of translation of natural mRNAs in an optimized in vitro system. , 1996, Archives of biochemistry and biophysics.

[13]  A. Plückthun,et al.  In vitro selection and evolution of functional proteins by using ribosome display. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[14]  A. Spirin,et al.  Direct expression of PCR products in a cell‐free transcription/translation system: synthesis of antibacterial peptide cecropin , 1997, FEBS letters.

[15]  F. Hartl,et al.  Recombination of protein domains facilitated by co-translational folding in eukaryotes , 1997, Nature.

[16]  H. Hayashi,et al.  NMR backbone assignments of the cyanobacterial transcriptional factor, SmtB, that senses the zinc concentration in the cell , 1998 .

[17]  R. Parker,et al.  The 3′ to 5′ degradation of yeast mRNAs is a general mechanism for mRNA turnover that requires the SKI2 DEVH box protein and 3′ to 5′ exonucleases of the exosome complex , 1998, The EMBO journal.

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

[19]  Y. Kobayashi,et al.  A pair of related genes with antagonistic roles in mediating flowering signals. , 1999, Science.

[20]  T. Sawasaki,et al.  A new class of enzyme acting on damaged ribosomes: ribosomal RNA apurinic site specific lyase found in wheat germ , 1999, The EMBO journal.

[21]  Thomas Szyperski,et al.  Protein NMR spectroscopy in structural genomics , 2000, Nature Structural Biology.

[22]  Y Endo,et al.  A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: plants apparently contain a suicide system directed at ribosomes. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[23]  R. Wickner,et al.  3' poly(A) is dispensable for translation. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[24]  E. V. Makeyev,et al.  Co-translational Folding of an Eukaryotic Multidomain Protein in a Prokaryotic Translation System* , 2000, The Journal of Biological Chemistry.

[25]  T. Muir,et al.  Protein engineering by expressed protein ligation. , 2000, Methods in enzymology.

[26]  H. Wang,et al.  Detection of a Novel Quiescence-dependent Protein Kinase* , 2000, The Journal of Biological Chemistry.

[27]  Anthony D. Keefe,et al.  The use of mRNA display to select high-affinity protein-binding peptides , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[28]  Masahiro Kasahara,et al.  Arabidopsis nph1 and npl1: Blue light receptors that mediate both phototropism and chloroplast relocation , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[29]  J. Vervoort,et al.  Role of Threonines in the Arabidopsis thaliana Somatic Embryogenesis Receptor Kinase 1 Activation Loop in Phosphorylation* , 2001, The Journal of Biological Chemistry.

[30]  D. Gallie.  Translational control of cellular and viral mRNAs , 1996, Plant Molecular Biology.