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Cell-Free Expression

A rapid method for gene expression analysis, PURExpress® is a novel cell-free transcription/translation system reconstituted from the purified components necessary for E. coli translation.

PURExpress® is a registered trademark of New England Biolabs, Inc.

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Protocols for Cell-Free Expression

Application Notes for Cell-Free Expression

    Publications related to Cell-Free Expression

  1. Iizuka, R., Yamagishi-Shirasaki, M. and Funatsu, T. 2011. Kinetic study of de novo chromophore maturation of fluorescent proteins Anal. Biochem. .  Apr 1. , PubMedID: 21459075 , DOI:
  2. Midon, M., Schäfer, P., Pingoud, A., Ghosh, M., Moon, A.F., Cuneo, M.J., London, R.E. and Meiss, G. 2011. Mutational and biochemical analysis of the DNA-entry nuclease EndA from Streptococcus pneumoniae Nucleic Acids Res. . 39, PubMedID: 20846957, DOI:
  3. Feng, Z., et al. 2011. Optimization of Medium Composition for Production of Recombinant Calf Chymosin from Kluyveromyces lactis in Submerged Fermentation J. of Northeast Agricultural University. 18, PubMedID: , DOI:
  4. Yamamoto, S., Izumiya, H., Mitobe, J., Morita, M., Arakawa, E., Ohnishi, M. and Watanabe, H. 2011. Identification of a chitin-induced small RNA that regulates translation of the tfoX gene, encoding a positive regulator of natural competence in Vibrio cholerae J Bacteriol. . 193, PubMedID: 21317321, DOI:
  5. De Masi, F., Grove, C.A., Vedenko, A., Alibés, A., Gisselbrecht, S.S., Serrano, L., Bulyk, M.L. and Walhout, A.J. 2011. Using a structural and logics systems approach to infer bHLH-DNA binding specificity determinants Nucleic Acids Res. . 18-Feb , PubMedID: 21335608 , DOI:
  6. Handa, Y., Inaho, N. and Nameki, N. 2011. YaeJ is a novel ribosome-associated protein inEscherichia coli that can hydrolyze peptidyl-tRNA on stalled ribosomes Nucleic Acids Res.  . 39, PubMedID: 21051357, DOI:
  7. Panayiotou, C., Solaroli, N., Xu, Y., Johansson, M. and Karlsson, A. 2011. The characterization of human adenylate kinases 7 and 8 demonstrates differences in kinetic parameters and structural organization among the family of adenylate kinase isoenzymes Biochem J.  . 2433 , PubMedID: 21080915, DOI:
  8. Lamichhane, T.N., Abeydeera, N.D., Duc, A.C., Cunningham, P.R. and Chow, C.S 2011. Selection of peptides targeting helix 31 of bacterial 16S ribosomal RNA by screening M13 phage-display libraries Molecules . 16, PubMedID: 21278676, DOI:
  9. Rosner, K., Kasprzak, M.F., Horenstein, A.C., Thurston, H.L., Abrams, J., Kerwin, L.Y., Mehregan, D.A. and Mehregan, D.R. 2011. Engineering a waste management enzyme to overcome cancer resistance to apoptosis: adding DNase1 to the anti-cancer toolbox Cancer Gene Ther. . 18, PubMedID: 21233855, DOI:
  10. Narayan, V., Pion, E., Landré, V., Müller, P. and Ball, K.L. 2011. Docking-dependent ubiquitination of the interferon regulatory factor-1 tumor suppressor protein by the ubiquitin ligase CHIP J. Biol. Chem. . 286, PubMedID: 20947504, DOI:
  11. Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K. and Ueda, T. 2001. Cell-free translation reconstituted with purified components Nat. Biotech.. 19, PubMedID: 11479568, DOI:
  12. Kaiser, C.M., Chang, H.C., Agashe, V.R., Lakshmipathy, S.K., Etchells, S.A., Hayer-Hartl, M., Hartl, F.U. and Barral, J.M. 2006. Real-time observation of trigger factor function on translating ribosomes Nature. 444, PubMedID: 17051157, DOI:
  13. Shimizu, Y. and Ueda, T. 2006. SmpB triggers GTP hydrolysis of elongation factor Tu on ribosome by compensating for the lack of codon-anticodon interaction during trans-translation initiation J. Biol. Chem.. 281, PubMedID: 16601123, DOI:
  14. Muto, H., Nakatogawa, H., and Ito, K. 2006. Genetically Encoded but Nonpolypeptide Prolyl-tRNA Functions in the A Site for SecM-Mediated Ribosomal Stall Mol. Cell.. 22, PubMedID: 16713584, DOI:
  15. Villemagne, D., Jackson, R. and Douthwaite, J.A. 2006. Highly efficient ribosome display selection by use of purified components for in vitro translation J. Immunol Methods. 313, PubMedID: 16730021, DOI:
  16. Shimizu, Y. Kanamori, T. and Ueda, T. 2005. Protein synthesus by pure translation systems Methods. 36, PubMedID: 16076456, DOI:
  17. Ying, B.W., Taguchi, H., Kondo, M. and Ueda, T. 2005. Co-translational involvement of the chaperonin GroEL in the folding of newly translated polypeptides J. Biol. Chem.. 280, PubMedID: 15664980, DOI:
  18. Kuruma, Y., Nishiyama, K., Shimizu, Y., Muller, M and Ueda, T. 2005. Development of a minimal cell-free translation system for the synthesis of presecretory and integral membrance proteins Biotechnol, Prog.. 21, PubMedID: 16080708, DOI:
  19. Sando, S., Kanatani, K., Sato, N., Matsumoto, H. Hohsaka, T. and Aoyama, Y. 2005. A small-molecule-based approach to sense codon-templated natural-unnatural hybrid peptides. Selective silencing and reassignment of the sense codon by orthogonal reacylation stalling at the single-codon level J. Am. Chem. Soc.. 127, PubMedID: 15926808, DOI:
  20. Theerthagiri, G., Eisenhardt, N., Schwarz, H. and Antonin, W. 2010. The nucleoporin Nup188 controls passage of membrane proteins across the nuclear pore complex  J. Cell Biol.  . 189, PubMedID: 20566687, DOI:
  21. Noto, T., Kurth, H., Kataoka, K., Aronica, L., DeSouza, L., Siu, K., Pearlman, R., Gorovsky, M. and Mochizuki, K. 2010. The tetrahymena argonaute-binding protein Giw1p directs a mature argonaute-siRNA complex to the nucleus Cell . 140 , PubMedID: 20211138, DOI:
  22. Shen, B.W., Heiter, D.F., Chan, S.H., Wang, H., Xu, S.Y., Morgan, R.D., Wilson, G.G. and Stoddard, B.L. 2010. Unusual target site disruption by the rare-cutting HNH restriction endonuclease PacI Structure . 18, PubMedID: 20541511, DOI:
  23. Ueda, T., Kanamori, T., Ohashi, H. 2010. Ribosome display with the PURE technology Methods Mol Biol. . 607, PubMedID: 20204860, DOI:
  24. Asahara, H. and Chong, S. 2010. In vitro genetic reconstruction of bacterial transcription initiation by coupled synthesis and detection of RNA polymerase holoenzyme Nucleic Acids Res. . 38, PubMedID: 20457746, DOI:
  25. Murtas, G., Kuruma, Y., Bianchini, P., Diaspro, A., and Luisi, P.L. 2007. Protein synthesis in liposomes with a minimal set enzymes Biochem. Biophys. Res. Commun.  . 363, PubMedID: 17850764, DOI:
  26. Ohta, A., Murakami, H., Higashimura, E., and Suga, H. 2007. Synthesis of Polyester by Means of Genetic Code Reprogramming Chem. Biol. . 14, PubMedID: 18096500, DOI:
  27. Sharma, C.M., Darfeuille, F., Plantinga, T.H., and Vogel, J. 2007. A small RNA regulates multiple ABC transporter mRNA's by targeting C/A-rich elements inside and upstream of ribosome-binding sites Gene Dev.. 21, PubMedID: 17974919, DOI:
  28. Subtelny. A. O.,Hartman, M. C. T., and Szostak, J. W. 2008. Ribosomal Synthesis of N-Methyl Peptides  J. Am. Chem. Soc. . 130, PubMedID: 18402453, DOI:
  29. Sako, Y., Goto, Y., Murakami, H., and Suga, H. 2008. Ribosomal Synthesis of Peptidase-Resistant Peptides Closed by a Nonreducible Inter-Side-Chain Bond ACS Chem. Biol.  . 3, PubMedID: 18338852, DOI:
  30. Vazquez-Laslop, N., Thum, C., and Mankin, A.S. 2008. Molecular Mechanism of Drug-Dependent Ribosome Stalling  Mol. Cell. . 30, PubMedID: 18439898, DOI:
  31. Uemura, S., Iizuka, R., Ueno, T., Shimizu, Y., Taguchi, H., Ueda, T., Puglisi. J., and Funatsu, T. 2008. Single molecule imaging of full protein synthesis by immobilized ribosomes Nuc. Acid. Res.  . 36, PubMedID: 18511463, DOI:
  32. Kazuta, Y., Adachi, J., Matsuura, T., Ono, N., Mori, H. and Yomo T. 2008. Comprehensive Analysis of the Effects of Eschericia coli ORF's on Protein Translation Reaction Molecular & Cellular Proteomics . 7, PubMedID: 18453339, DOI:
  33. Estevez-Torres, A., Crozatier, C., Diguet, A., Hara, T., Saito, H., Yoshikawa, K., and Baigl, D. 2009. Sequence-independent and reversible photocontrol of transcription/expression systems using a photosensitive nucleic acid binder Proc. Natl. Acad. Sci.  . 106, PubMedID: 19617550, DOI:
  34. Talabot-Ayer, D., Lamacchia, C., Gabay, C., and Palmer, G. 2009. Interleukin-33 is biologically active independently of Caspase-1 cleavage J. Biol. Chem. . 284, PubMedID: 19465481, DOI:
  35. Solaroli, N., Panayiotou, C., Johansson, M., and Karlsson, A. 2009. Identification of two active functional domains of human adenylate kinase 5 FEBS Lett. . 583, PubMedID: 19647735, DOI:
  36. Zheng, Y., Posfai, J., Morgan, R. D., Vincze, T., and Roberts, R.J. 2009. Using shotgun sequence data to find active restriction enzyme genes Nuc. Acid. Res. . 37, PubMedID: 18988632, DOI:
  37. Feng, Y. and Cronan, J. E. 2009. A new member of the Eschericia coli fad regulon: transcriptional regulation of fadM (ybaW) J. Bacteriol. . 191, PubMedID: 19684132, DOI:
  38. Tanner, D., Cariello, D., Woolstenhulme, C., Broadbent, M. and Buskirk, A. 2009. Genetic identification of nascent peptides that induce ribosome stalling J. Biol. Chem. . 284, PubMedID: 19840930, DOI:

The PURExpress® System Kit Components

  • His-Tagged Translation Factors
    • Initiation Factors (IF1, IF2, IF3)
    • Elongation Factors (EF-Tu, EF-Ts, EF-G)
    • Release Factors (RF1, RF2, RF3)
    • Ribosome Recycling Factor
    • 20 Aminoacyl tRNA synthetases
    • Methionyl tRNA formyltransferase
  • E. coli Ribosomes
  • E. coli tRNAs
  • Energy Regeneration System
  • NTPs, Amino Acids, Salts, Buffer

In addition, recombinant T7 RNA polymerase is used to couple transcription to translation. The PURE system represents an important step towards a totally defined in vitro transcription/translation system, thus avoiding the “black box” nature of the cell extract-based systems.

Protein Expression Using the PURExpress® In Vitro Protein Synthesis Kit

25 µl reactions containing 250 ng template DNA were incubated at 37°C for 2 hours. 2.5 µl of each reaction was analyzed by SDS-PAGE using a 10–20% Tris-glycine gel. Note that proteins can be purified using reverse affinity chromatography (reagents not supplied). The red dot indicates the protein of interest. Marker M is the Protein Ladder (NEB #P7703)