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  • Protein Expression

    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.

    Solutions for the Expression of Difficult Proteins

    NEB has a long history in recombinant protein expression and has developed a wide array of solutions for proteins that are difficult to express.

    NEB has a long history in recombinant protein expression and has developed a wide array of solutions for proteins that are difficult to express.

      Publications related to Cell-Free Expression:

    1. 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, 751-755. PubMedID: 11479568
    2. 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, 7998-7999. PubMedID: 15926808
    3. 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, 12035-12040. PubMedID: 15664980
    4. 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, 1243-1251. PubMedID: 16080708
    5. Shimizu, Y. Kanamori, T. and Ueda, T. (2005) Protein synthesus by pure translation systems Methods 36, 299-304. PubMedID: 16076456
    6. 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, 545-552. PubMedID: 16713584
    7. 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, 140-148. PubMedID: 16730021
    8. 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, 455-460. PubMedID: 17051157
    9. 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, 15987-15996. PubMedID: 16601123
    10. 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, 2804-2817. PubMedID: 17974919
    11. 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, 12-17. PubMedID: 17850764
    12. Ohta, A., Murakami, H., Higashimura, E., and Suga, H. (2007) Synthesis of Polyester by Means of Genetic Code Reprogramming Chem. Biol. 14, 1315-1322. PubMedID: 18096500
    13. 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, 1530-1540. PubMedID: 18453339
    14. Vazquez-Laslop, N., Thum, C., and Mankin, A.S. (2008) Molecular Mechanism of Drug-Dependent Ribosome Stalling  Mol. Cell. 30, 190-202. PubMedID: 18439898
    15. Subtelny. A. O.,Hartman, M. C. T., and Szostak, J. W. (2008) Ribosomal Synthesis of N-Methyl Peptides  J. Am. Chem. Soc. 130, 6131-6136. PubMedID: 18402453
    16. 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, 241-249. PubMedID: 18338852
    17. 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, e90. PubMedID: 18511463
    18. Feng, Y. and Cronan, J. E. (2009) A new member of the Eschericia coli fad regulon: transcriptional regulation of fadM (ybaW) J. Bacteriol. 191, 6320-6328. PubMedID: 19684132
    19. 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, 34809-34818. PubMedID: 19840930
    20. Solaroli, N., Panayiotou, C., Johansson, M., and Karlsson, A. (2009) Identification of two active functional domains of human adenylate kinase 5 FEBS Lett. 583, 2872-2876. PubMedID: 19647735
    21. 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, 19420-19426. PubMedID: 19465481
    22. 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, 12219-12223. PubMedID: 19617550
    23. 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, el. PubMedID: 18988632
    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, e141. PubMedID: 20457746
    25. Ueda, T., Kanamori, T., Ohashi, H. (2010) Ribosome display with the PURE technology Methods Mol Biol. 607, 219-25. PubMedID: 20204860
    26. 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, 1129-1142. PubMedID: 20566687
    27. 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, 692-703. PubMedID: 20211138
    28. 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, 734-743. PubMedID: 20541511
    29. 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, 607-619. PubMedID: 20947504
    30. 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, 346-357. PubMedID: 21233855
    31. 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, 1953-1965. PubMedID: 21317321
    32. 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
    33. 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, 623-634. PubMedID: 20846957
    34. 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, 56-62.
    35. 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, 1211-1239. PubMedID: 21278676
    36. 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, 527-534. PubMedID: 21080915
    37. 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
    38. 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, 1739-1748. PubMedID: 21051357

    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)