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PURExpress™ In Vitro Protein Synthesis Kit |
 | | | | PURExpress™ In Vitro Protein Synthesis Kit |
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Prices are in US dollars and valid only for US orders.
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- 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
Description:
PURExpress is a novel coupled cell-free transcription/translation system reconstituted from purified components necessary for E. coli translation. Recombinant histidine-tagged aminoacyl-tRNA synthetases (20), initiation factors (3), elongation factors (3), release factors (3), ribosome recycling factor, methionyl-tRNA transformylase, T7 RNA polymerase, creatine kinase, myokinase, nucleoside-di-phosphate kinase, and pyrophophatase provide the activities required for coupled transcription and translation and energy regeneration. Purified 70S ribosomes, amino acids, rNTP’s, and tRNA’s complete the system. NEB currently offers two PURExpress kits: in #E6800 all the components are contained in two solutions (A&B); in #E3313 the ribosomes are added to the reaction separately.
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). PURExpress is an easy-to-use one-step reaction that requires the mixing of only two tubes. Protein synthesis is initiated by the addition of template DNA and is largely complete within two hours. Products of translation can be analyzed by SDS-PAGE (Coomassie stained, autoradiograph of 35S-labeled proteins, or western blot) or in direct activity assays. Purification of the target protein can often be accomplished by ultrafiltration to remove the high MW ribosomes followed by IMAC (immobilized metal affinity chromatography) to remove the His-tagged components.
Due to its reconstitution of recombinant components, PURExpress is essentially free of contaminating exonucleases, RNases, and proteases. Template DNA is not exposed to digestion and target proteins are free of post-translational modifications (glycosylation, phosphorylation, and proteolysis).
Notice to PURExpress Customers:
Effective December 1, 2009, the PURExpress In vitro Protein Synthesis Kit (NEB #E6800) will be offered with a revised formulation and more convenient reaction format.
The new reaction format still utilizes a 25 μl standard reaction volume. The amounts of solutions A and B have been changed to 10 and 7.5 μl, to be added respectively. The new formulation allows an increase in the "user-added" amount as well, tolerating up to 20% over volume without an appreciable drop in productivity. The final concentration of each amino acid remains 0.3 mM in a 25 μl reaction.
Other changes to the system include a revised control DHFR template. The backbone of this vector has been modified to be a high copy plasmid with a T7 terminator downstream of the DHFR coding sequence. This change creates a control plasmid that can also be utilized as a cloning vector for target genes. The addition of the terminator sequence prevents wasteful transcription and often results in increases in translation efficiency. Users can replace the DHFR gene with other genes of interest for use both in vitro or in vivo.
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).
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
Kit Components:
Control (DHFR) template (10 μl)
Solution A (100 μl)
Solution B (75 μl)
Universal Primer (25 μl)
Storage Conditions
Storage Temperature:
-80°C
Notes
General notes:- PURExpress DHFR Control Template sequence files: Fasta GenBank
PURExpress Universal Primer sequence files: Fasta GenBank - Storage: All kit components should be stored at -80°C.
Usage notes:- The DHFR control template is now supplied at 125 ng/µl. Use 2 µl for the 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.
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 25–250 ng template per 25 μl reaction.
FAQs
- Where can I find many more detailed FAQs for PURExpress?
- When using PURExpress, I was unable to synthesize the control protein?
- When using PURExpress, I was able to synthesize the control protein, but the target sample is not present or present in low yield?
- When using PURExpress, I was able to synthesize the target protein, but full-length product is not major species?
Protocols
Protocols for PURExpress™ In Vitro Protein Synthesis Kit
References
- 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.
- 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.
- 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.
Companion Products
Murine RNase Inhibitor
PURExpress™ Δ Ribosome Kit
Legal
Licenses/Patents/Disclaimers:
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™ Δ Ribosome Kit requires a license from New England Biolabs, Inc.
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New
England Biolabs, Inc. is an ISO 9001 and ISO
14001 Certified Company.
NEB certifies that it is a small business in accordance with the US Small Business Administration and 13 CFR 121.201 |
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