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
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.
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. 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, discontinued and replaced with NEB #P7717 ).
This product is related to the following categories:
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.
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.
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.
PURExpress DHFR Control Template sequence files: Fasta, GenBank
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.
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
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
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
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
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
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
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
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
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
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
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
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
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
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.
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
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
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
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.
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
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 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.
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]
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]
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.
Licenses
PURExpress® is based on the PURE System Technology originally developed by Dr. Takuya Ueda at the University of Tokyo and commercialized as the PURESYSTEM® by BioComber (Tokyo, Japan).
Licensed from BioComber (Tokyo, Japan) under Patent Nos. 7,118,883; WO2005-105994 and JP2006-340694. For research use only. Commercial use of PURExpress® In vitro Protein Synthesis Kit requires a license from New England Biolabs, Inc. 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.
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