Product Class: Kit

PURExpress® Δ (aa, tRNA) Kit

Product Introduction

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
Catalog # Size Concentration
E6840S 10.0 reactions

Product Information

Description

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

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 (75 μl) P0760AVIAL -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

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

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. 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. 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. 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. 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. 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. 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. 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. 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), 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. 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. 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. 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. 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. 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. 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. PubMedID: 28754692

Protocols, Manuals & Usage

Protocols

  1. Protein Synthesis Reaction using PURExpress® ∆ (aa, tRNA) Kit (E6840)
  2. Analysis of Synthesized Protein using PURExpress (E6840)
  3. Measurement of 35S-Methionine Incorporation by TCA Precipitation and Yield Determination using PURExpress
  4. Determination of Protein Synthesis Yield with PURExpress (E3313, E6800, E6840, E6850)
  5. Purification of Synthesized Protein using Reverse His-tag Purification

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Application Notes

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. How is the Δ aa Δ tRNA Kit E6840S different from the PURExpress E6800S kit?
  2. When using PURExpress, I was unable to synthesize the control protein?
  3. When using PURExpress, I was able to synthesize the target protein, but full-length product is not major species?
  4. When using PURExpress, I was able to synthesize the control protein, but the target sample is not present or present in low yield?
  5. Where can I find many more detailed FAQs for PURExpress?
  6. Are there PURExpress citations?

Tech Tips

Thaw and assemble reactions on ice
Thoroughly mix solutions A and B before using. Do not vortex Solution B or ribosomes, mix gently.
Solution A may have a cloudy white appearance. Add to the reaction as a uniform suspension.
Assemble the reactions in the following order on ice: Solution A, Solution B, RNAse Inhibitor, Water, Template DNA or RNA
Once reaction is assembled take time to make sure everything is thoroughly mixed by gently pipetting up and down, pulse spin and place at 37C for 2 to 4 hours.

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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