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  • PURExpress® Δ Ribosome Kit

    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. The relative nuclease-free and protease-free nature of the PURExpress platform 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.
    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 ).
    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 4: Expression and reverse purification of DHFR (A) and T4 DNA Ligase (B) using PURExpress.
    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 ).

    Highlights

    • Cleaner System - sample degradation eliminated
    • Easy-to-use - protein expression complete in approximately two hours
    • Simple Analysis - protein can often be visualized directly on a Coomassie stained gel

    Kit Components

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Factor Mix
    E. coli Ribosome-80
    PURExpress Solution A
    Control (DHFR) template

    Advantages and Features

    Applications

    • 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

    Properties and 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

    Storage Temperature

    -80°C

    Notes

    1. The DHFR control template is now supplied at 125 ng/µl. Use 2 µl for the positive control reaction. We use 60 pmoles of ribosomes in a standard 25 μl reaction. The supplied control ribosomes are enough for two reactions. If your ribosome concentration is more than 13.3 μM, we suggest you make up the ribosome volume to 4.5 μl with 10 mM magnesium acetate. Note: Using a smaller amount of ribosomes is possible but the protein yield may be lower.  For detailed usage information please refer to the product manual.
    2. PURExpress Control Template sequence files: Fasta, GenBank
    3. Storage: All kit components should be stored at -80°C.

    References

    1. Gupta, P., K. Kannan, et al. (2013). Regulation of Gene Expression by Macrolide-Induced Ribosomal Frameshifting. Mol Cell. 52(5), 629-42. PubMedID: 24239289
    2. Gupta, P., S. Sothiselvam, et al. (2013). Deregulation of translation due to post-transcriptional modification of rRNA explains why erm genes are inducible. Nat Commun . 4, 1984. PubMedID: 23749080
    3. Harvey, C. J., J. D. Puglisi, et al. (2012). Precursor directed biosynthesis of an orthogonally functional erythromycin analogue: selectivity in the ribosome macrolide binding pocket. J Am Chem Soc. 134(29), 12259-65. PubMedID: 22741553
    4. Kaiser, C. M., D. H. Goldman, et al. (2011). The ribosome modulates nascent protein folding. Science. 334(6063), 1723-7. PubMedID: 22194581
    5. Kannan, K., N. Vázquez-Laslop, et al. (2012). Selective Protein Synthesis by Ribosomes with a Drug-Obstructed Exit Tunnel. Cell. 151(3), 508-520. PubMedID: 23101624
    6. Kopaskie, K. S., K. G. Ligtenberg, et al. (2013). Translational regulation of Yersinia enterocolitica mRNA encoding a type III secretion substrate. Journal of Biological Chemistry. 288(49), 35478-88. PubMedID: 24158443
    7. 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
    8. Orelle, C., S. Carlson, et al. (2013). Tools for Characterizing Bacterial Protein Synthesis Inhibitors. Antimicrob Agents Chemother. 57(12), 5994-6004. PubMedID: 24041905
    9. Shi, W., X. Zhang, et al. (2011). Pyrazinamide inhibits trans-translation in Mycobacterium tuberculosis. Science. 333(6049), 1630-1632. PubMedID: 21835980
    10. Tsai, A., J. Chen, et al. (2013). Observing Prokaryotic Translation Elongation in Real-Time using Single-Molecule Fluorescence. Biophysical Journal. 104(2, Supplement 1), 257a.
    11. Vazquez-Laslop, N., H. Ramu, et al. (2010). The key function of a conserved and modified rRNA residue in the ribosomal response to the nascent peptide. EMBO J. 29(18), 3108-3117. PubMedID: 20676057
    12. Vázquez-Laslop, N., H. Ramu, et al. (2011). Nascent peptide-mediated ribosome stalling promoted by antibiotics. Ribosomes. 377-392.

    FAQs

    1. Detailed FAQs for PURExpress?
    2. How is the Δ Ribosome Kit E3313S different from the PURExpress E6800S kit?
    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 unable to synthesize the control protein?
    5. When using PURExpress, I was able to synthesize the control protein, but the target sample is not present or present in low yield?
    6. IMPACT FAQs
    7. 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.

    Protocols

    1. Protein Synthesis Reaction using PURExpress (E3313)
    2. Analysis of Synthesized Protein using PURExpress (E3313)
    3. Determination of Protein Synthesis Yield with PURExpress (E3313)
    4. Purification of Synthesized Protein using Reverse His-tag Purification
    5. Measurement of 35S-Methionine Incorporation by TCA Precipitation and Yield Determination using PURExpress

    Manuals

    The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

    Safety Data Sheet

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

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.