Home Protein Expression Products PURExpress® Δ (aa, tRNA) Kit

PURExpress® Δ (aa, tRNA) Kit

PURExpress® Δ (aa, tRNA) Kit is a variation of the PURExpress In vitro Protein Synthesis Kit where the amino acids and tRNA are omitted from the translation mix.

  • Control amino acids and tRNA are provided separately
  • Ideal for studies requiring control of the concentration or content of amino acids and/or tRNAs in a translation reaction
  • Applications include non-natural amino acid incorporation, amino acid tRNA synthetase studies and labeling with amino acids that contain stable isotopes
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Catalog # Concentration Size List Price Your Price Quantity
E6840S Not Applicable 10 reactions $408.00
Please enter a quantity for at least one size

  • 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. With minimal nuclease and protease activity, the PURExpress system preserves the integrity of DNA and RNA templates/complexes and results in proteins that are free of modification and degradation. Transcription and translation are carried out in a one-step reaction, and require the mixing of only two tubes. With results available in a few hours, PURExpress saves valuable laboratory time and is ideal for high throughput technologies.


    PURExpress Citations


    Figure 1: Protein expression using the PURExpress® In Vitro Protein Synthesis Kit. Figure 1: Protein expression using the PURExpress® In Vitro Protein Synthesis Kit.

    25 μl reactions containing 250 ng template DNA and 20 units RNase Inhibitor 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. The red dot indicates the protein of interest. Marker M is the Protein Ladder (NEB #P7703, discontinued and replaced with NEB #P7717 ).

    Figure 2: Incorporation of 35S-methionine enables visualizationof protein by autoradiography.
    Figure 2: Incorporation of 35S-methionine enables visualizationof protein by autoradiography.
    25 μl reactions containing 250 ng template DNA, 20 units RNase Inhibitor and 2 μl 35S-met were incubated at 37°C for 2 hours. 2.5 μl of each reaction was analyzed by SDS-PAGE, the gel was fixed for 10 minutes, dried for 2 hours at 80°C and exposed to x-ray film for 5 hours at -80°C.

    Figure 3: Schematic diagram of protein synthesis and purification by PURExpress. Figure 3: Schematic diagram of protein synthesis and purification by PURExpress.

    Figure 4: Expression and reverse purification of DHFR (A) and T4 DNA Ligase (B) using PURExpress. Figure 4: Expression and reverse purification of DHFR (A) and T4 DNA Ligase (B) using PURExpres

    125 μl reactions were carried out according to recommendations in the accompanying manual. Samples were analyzed on a 10–20% Tris-glycine gel and stained with Coomassie Blue. Note that in both cases, the desired protein can be visualized in the total protein fraction. The red dot indicates the protein of interest. Marker M is the Protein Ladder (NEB #P7703, discontinued and replaced with NEB #P7717 ).


    This product is related to the following categories:
    PURExpress,
    Cell-Free Protein Expression Products,
    Protein Expression Products
    This product can be used in the following applications:
    PURExpress,
    Cell-Free Protein Expression,
    Protein Expression

    • Kit Components

      The following reagents are supplied with this product:

      NEB # Component Name Component # Stored at (°C) Amount Concentration
      • E6840S     -80    
          PURExpress Solution A (-aa, tRNA) B6841AVIAL -80 1 x 0.05 ml 5 X
          E. coli tRNA (25 μl) N6842AVIAL -80 1 x 0.025 ml Not Applicable
          Amino Acid Mix (25 μl) N6843AVIAL -80 1 x 0.025 ml Not Applicable
          Solution B P6840AVIAL -80 1 x 0.075 ml Not Applicable
          PURExpress Control DHFR Plasmid N0424AVIAL -20 1 x 0.01 ml 125 ng/µl

    • Properties & Usage

      Materials Required but not Supplied

      • General:  37°C incubator
      • Labeling:  35S-Methionine (>1000 Ci/mmol recommended, in vitro translation grade)
      • TCA Precipitation:  TCA solutions (25%, 10%), 1 M NaOH, casamino acids, ethanol, glass fiber filters, vacuum filtration manifold
      • SDS-PAGE:  Gels and running buffer, gel apparatus, power supply, gel dryer
      • Western Blotting:  Transfer apparatus, membrane, antibodies and detection reagent
      • Purification:  Ni-NTA Agarose, Amicon Ultra- 0.5 ml, Ultracel- 100K Membrane Centrifugal Filters

    • Advantages and Features

      Application Features

      • Quickly generate analytical amounts of protein for further characterization
      • Confirmation of open reading frames
      • Examination of the effects of mutations on ORFs
      • Generation of truncated proteins to identify active domains and functional residues
      • Introduction of modified, unnatural or labeled amino acids
      • Epitope mapping
      • Expression of toxic proteins
      • Ribosome display
      • Translation and/or protein folding studies
      • In vitro compartmentalization

    • Related Products

      Companion Products

    • Product Notes

      1. For a positive control reaction, use 2 μl of the supplied DHFR control template and 2.5 μl each of the supplied aa and tRNA.
      2. Each kit contains sufficient reagents for 10 x 25 µl reactions.
        The amino acids and tRNA are supplied separately, allowing users to perform a protein synthesis reaction by adding modified amino acids and tRNA mixture to the reaction.
      3. Add Solution B to Solution A, do not dilute Solution B unbuffered. We recommend a starting concentration of 250 ng template DNA per 25 μl reaction. The optimal amount of input DNA can be determined by setting up multiple reactions and titrating the amount of template DNA added to the reaction. Typically, the optimal amount will fall in a range of 25-1000 ng template per μl reaction.
      4. PURExpress DHFR Control Template sequence files: Fasta, GenBank
      5. The DHFR control template is now supplied at 125 ng/μl. Use 2 μl for a positive control reaction. Template DNA, particularly plasmid DNA prepared by mini-prep (e.g. Qiagen) is often the major source of RNase contamination. We strongly recommend adding 20 units Murine RNase Inhibitor  (NEB #M0314) to each reaction.

    • References

      1. Martínez, A. K., E. Gordon, et al. (2013). Interactions of the TnaC nascent peptide with rRNA in the exit tunnel enable the ribosome to respond to free tryptophan. Nucleic Acids Research. 42(2), 1245-56. PubMedID: 24137004
      2. Horiya S, Bailey J.K., Krauss I.J. (2017). Directed Evolution of Glycopeptides Using mRNA Display.. Methods in Enzymology. DOI: 10.1016/bs.mie.2017.06.029 PubMedID: 28935113
      3. Houwman J.A., André E, Westphal A.H., van Berkel W.J., van Mierlo C.P. (2016). The Ribosome Restrains Molten Globule Formation in Stalled Nascent Flavodoxin.. The Journal of Biological Chemistry. 291 (50), 25911-25920. DOI: 10.1074/jbc.M116.756205 PubMedID: 27784783
      4. Huang W.P., Cho C.P., Chang K.Y. (2018). mRNA-Mediated Duplexes Play Dual Roles in the Regulation of Bidirectional Ribosomal Frameshifting.. International Journal of Molecular Sciences. 19 (12), 3867. DOI: 10.3390/ijms19123867 PubMedID: 30518074
      5. Martínez A.K., Gordon E, Sengupta A, Shirole N, Klepacki D, Martinez-Garriga B, Brown L.M., Benedik M.J., Yanofsky C, Mankin A.S., Vazquez-Laslop N, Sachs M.S., Cruz-Vera L.R. (2014). Interactions of the TnaC nascent peptide with rRNA in the exit tunnel enable the ribosome to respond to free tryptophan.. Nucleic Acids Research. 42 (2), 1245-1256. DOI: 10.1093/nar/gkt923 PubMedID: 24137004
      6. Seefeldt A.C., Graf M, Perebaskine N, Nguyen F, Arenz S, Mardirossian M, Scocchi M, Wilson D.N., Innis C.A. (2016). Structure of the mammalian antimicrobial peptide Bac7(1–16) bound within the exit tunnel of a bacterial ribosome. Nucleic Acids Research. 44 (5), 2429-2438. DOI: 10.1093/nar/gkv1545 PubMedID: 26792896
      7. Seefeldt A.C., Nguyen F., Antunes S, Perebaskine N, Graf M, Arenz S, Inampudi K.K., Douat C, Guichard G, Wilson D.N., Innis C.A. (2015). The proline-rich antimicrobial peptide Onc112 inhibits translation by blocking and destabilizing the initiation complex.. Nature Structural and Molecular Biology. 22 (6), 470-475. DOI: 10.1038/nsmb.3034 PubMedID: 25984971
      8. Starosta A.L., Lassak J, Peil L, Atkinson G.C., Virumäe K, Tenson T, Remme J, Jung K, Wilson D.N. (2014). Translational stalling at polyproline stretches is modulated by the sequence context upstream of the stall site.. Nucleic Acids Research. 42 (16), 10711-10719. DOI: 10.1093/nar/gku768 PubMedID: 25143529
      9. Ude S, Lassak J, Starosta A.L., Kraxenberger T, Wilson D.N., Jung K (2013). Translation elongation factor EF-P alleviates ribosome stalling at polyproline stretches.. Science. 339 (6115), 82-85. DOI: 10.1126/science.1228985 PubMedID: 23239623
      10. Wensel D, Sun Y, Li Z, Zhang S, Picarillo C, McDonagh T, Fabrizio D, Cockett M, Krystal M, Davis J (2017). Discovery and Characterization of a Novel CD4-Binding Adnectin with Potent Anti-HIV Activity.. Antimicrobial Agents and Chemotherapy. 61 (8), DOI: 10.1128/AAC.00508-17 PubMedID: 28584151
      11. Xian F, Li S, Liu S (2015). Rapid biosynthesis of stable isotope-labeled peptides from a reconstituted in vitro translation system for targeted proteomics.. Methods in Enzymology. 565, 347-366. DOI: 10.1016/bs.mie.2015.07.013 PubMedID: 26577738
      12. Ude, S., J. Lassak, et al. (2013). Translation Elongation Factor EF-P Alleviates Ribosome Stalling at Polyproline Stretches. Science. 339(6115), 82-5. PubMedID: 23239623
      13. Zukher I, Novikova M, Tikhonov A, Nesterchuck M.V., Osterman I.A., Djordjevic M, Sergiev P.V., Sharma C.M., Severinov K (2014). Ribosome-controlled transcription termination is essential for the production of antibiotic microcin C.. Nucleic Acids Research. 42 (19), 11891-11902. DOI: 10.1093/nar/gku880 PubMedID: 25274735
      14. Arenz S, Bock L.V., Graf M, Innis C.A., Beckmann R, Grubmüller H, Vaiana A.C., Wilson D.N. (2016). A combined cryo-EM and molecular dynamics approach reveals the mechanism of ErmBL-mediated translation arrest.. Nature Communications. 7, 12026. DOI: 10.1038/ncomms12026 PubMedID: 27380950
      15. Cui Z, Stein V, Tnimov Z, Mureev S, Alexandrov K (2015). Semisynthetic tRNA Complement Mediates in vitro Protein Synthesis. Journal of the American Chemical Society. 137 (13), 4404-4413.
      16. Doerfel L.K., Wohlgemuth I, Kubyshkin V, Starosta A.L., Wilson D.N., Budisa N, Rodnina M.V. (2015). Entropic Contribution of Elongation Factor P to Proline Positioning at the Catalytic Center of the Ribosome. Journal of the American Chemical Society. 137 (40), 12997-13006. DOI: 10.1021/jacs.5b07427 PubMedID: 26384033
      17. Fleming S.R., Bartges T.E., Vinogradov A.A., Kirkpatrick C.L., Goto Y, Suga H, Hicks L.M., Bowers, A.A. (2019). Flexizyme-Enabled Benchtop Biosynthesis of Thiopeptides.. Journal of the American Chemical Society. 141 (2), 758-762. DOI: 10.1021/jacs.8b11521 PubMedID: 30602112
      18. Gan Q, Fan C Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis. Biochimica et Biophysica Acta - General Subjects. 3047-3052. DOI: 10.1016/j.bbagen.2016.12.002 PubMedID: 27919800
      19. Glover W.B., Mash D.C., Murch S.J. (2014). The natural non-protein amino acid N-beta-methylamino-L-alanine (BMAA) is incorporated into protein during synthesis. Amino Acids. 46 (11), 2553-2559.
      20. Hamadani K.M., Howe J, Jensen M.K., Wu P, Cate J.H.D., Marquee S (2017). An in vitro tag-and-modify protein sample generation method for single-molecule fluorescence resonance energy transfer.. The Journal of Biological Chemistry. 292 (38), 15636-15648. DOI: 10.1074/jbc.M117.791723 PubMedID: 28754692

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