Home › Products › PURExpress® In Vitro Protein Synthesis Kit
PURExpress® In Vitro Protein Synthesis Kit
Catalog #
E6800S
E6800L
Size
10 reactions
100 reactions
Concentration
Categories: Cell-Free Expression
Applications: Cell-Free Protein Expression, Disulfide-Bonded Protein Expression, Toxic Protein Expression
Product
Information
FAQs &
Tech Tips
Description
Properties and Usage
Notes
Protocols &
Manuals
Other Tools &
Resources
Quality &
Safety
Legal
Information
Advantages and Features
Related Products
References
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
PURExpress Solution A
Control (DHFR) template
PURExpress Solution B
3.3X
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
Storage Temperature
-80°C
Related Products
Companion Products
PURExpress® Δ (aa, tRNA) Kit
PURExpress® Δ RF123 Kit
RNase Inhibitor, Murine
PURExpress® Δ Ribosome Kit
E. coli Ribosome
PURExpress® Disulfide Bond Enhancer
Notes
1. 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.
2. PURExpress DHFR Control Template sequence files: Fasta GenBank
3. Storage: All kit components should be stored at -80°C.
4. Add Solution B to Solution A, do not dilute Solution B unbuffered. We recommend a starting amount 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 25 μl reaction.
References
1. Asahara, H. and Chong, S. (2010). In vitro genetic reconstruction of bacterial transcription initiation by coupled synthesis and detection
of RNA polymerase holoenzyme. Nuc. Acid. Res.
2. Noto, T., Kurth, H., Kataoka, K., Aronica, L., DeSouza, L., Siu, K., Pearlman, R., Gorovsky, M. and Mochizuki, K. (2010). The
tetrahymena argonaute-binding protein Giw1p directs a mature argonaute-siRNA complex to the nucleus. Cell. 140, 692-703.
3. Tanner, D., Cariello, D., Woolstenhulme, C., Broadbent, M. and Buskirk, A. (2009). Genetic identification of nascent peptides that
induce ribosome stalling. J. Biol. Chem. 284, 34809-34818.
4. 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.
5. 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.
6. Solaroli, N., Panayiotou, C., Johansson, M., and Karlsson, A. (2009). Identification of two active functional domains of human adenylate
kinase 5. FEBS Lett. 283, 2872-2876.
7. Arenz, Stefan, Haripriya Ramu, Pulkit Gupta, Otto Berninghausen, Roland Beckmann, Nora Vázquez-Laslop, Alexander S. Mankin,
and Daniel N. Wilson (2014). Molecular basis for erythromycin-dependent ribosome stalling during translation of the ErmBL leader
peptide. Nature communications. 5
8. Chong, Shaorong (2014). Overview of Cell Free Protein Synthesis: Historic Landmarks, Commercial Systems, and Expanding
Applications. Current Protocols in Molecular Biology. 16-30.
9. Daugherty, Ashley B., Sridhar Govindarajan, and Stefan Lutz (2013). Improved Biocatalysts from a Synthetic Circular Permutation
Library of the Flavin-Dependent Oxidoreductase Old Yellow Enzyme. Journal of the American Chemical Society . 334 (38), 1442514432.
10. Desai, Bijoy J., Yuki Goto, Alessandro Cembran, Alexander A. Fedorov, Steven C. Almo, Jiali Gao, Hiroaki Suga, and John A. Gerlt
(2014). Investigating the role of a backbone to substrate hydrogen bond in OMP decarboxylase using a site-specific amide to ester
substitution. Proceedings of the National Academy of Sciences. 201411772.
11. Gu, Liangcai, Chao Li, John Aach, David E. Hill, Marc Vidal, and George M. Church (2014). Multiplex single-molecule interaction
profiling of DNA-barcoded proteins. Nature.
12. Gupta, Pulkit, Shanmugapriya Sothiselvam, Nora Vázquez-Laslop, and Alexander S. Mankin (2013). Deregulation of translation due to
post-transcriptional modification of rRNA explains why erm genes are inducible. Nature communications. 4
13. Kaiser, Christian M., Daniel H. Goldman, John D. Chodera, Ignacio Tinoco, and Carlos Bustamante (2011). The ribosome modulates
nascent protein folding. Science. 334 (6063), 1723-1727.
14. Nakagawa, So, Stephen S. Gisselbrecht, Julia M. Rogers, Daniel L. Hartl, and Martha L. Bulyk (2013). DNA-binding specificity
changes in the evolution of forkhead transcription factors. Proceedings of the National Academy of Sciences. 110(30), 12349-12354.
15. Ramadoss, Nitya S., John N. Alumasa, Lin Cheng, Yu Wang, Sharon Li, Benjamin S. Chambers, Hoon Chang et al (2013). Small
molecule inhibitors of trans-translation have broad-spectrum antibiotic activity. Proceedings of the National Academy of Sciences.
110(25), 10282-10287.
16. Rosenblum, Gabriel, and Barry S. Cooperman (2014). Engine out of the chassis: Cell-free protein synthesis and its uses. FEBS letters.
588(2), 261-268.
17. Stafford, Ryan L., Marissa L. Matsumoto, Gang Yin, Qi Cai, Juan Jose Fung, Heather Stephenson, Avinash Gill et al (2014). In vitro
Fab display: a cell-free system for IgG discovery. Protein Engineering Design and Selection. 27(4), 97-109.
18. Tuckey, Corinna, Haruichi Asahara, Ying Zhou, and Shaorong Chong (2014). Protein Synthesis Using a Reconstituted Cell Free
System. Current Protocols in Molecular Biology. 16-31.
19. Weirauch, Matthew T., Atina Cote, Raquel Norel, Matti Annala, Yue Zhao, Todd R. Riley, Julio Saez-Rodriguez et al (2013). Evaluation
of methods for modeling transcription factor sequence specificity. Nature biotechnology. 31(2), 126-134.
FAQs
Tech Tips
FAQs
1. When using PURExpress, I was unable to synthesize the control protein?
2. When using PURExpress, I was able to synthesize the control protein, but the target sample is not present or present in low
yield?
3. When using PURExpress, I was able to synthesize the target protein, but full-length product is not major species?
4. Detailed FAQs for PURExpress?
5. 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
Datacards
Manuals
Protocols
1.
2.
3.
4.
5.
Protein Synthesis Reaction using PURExpress (E6800)
Analysis of Synthesized Protein using PURExpress (E6800)
Determination of Protein Synthesis Yield with PURExpress (E6800)
Purification of Synthesized Protein using Reverse His-tag Purification
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].
manualE6800
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 [email protected] or fill out the
Technical Support Form for appropriate document.
E6800Datasheet-Lot0161412
E6800Datasheet-Lot0171501
Selection Charts
Application Notes
Selection Charts
Protein Expression and Purification Selection Chart
Application Notes
Use of the PURExpress In Vitro Protein Synthesis Kit Disulfide Bond Enhancer and SHuffle Competent E coli for heterologous
in vitro and in vivo cellulase expression E6800
Using the PURExpress In Vitro ProteinSynthesis Kit for Heterologous In Vitro Expression and Functional Screening of FMN
dependent Oxidoreductase Variants E6800
Safety Data Sheet
Datacards
Safety Data Sheet
The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.
PURExpress Solution A
Control (DHFR) template (10 μl)
Solution B (75 μl)
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 [email protected] or fill out the
Technical Support Form for appropriate document.
E6800Datasheet-Lot0161412
E6800Datasheet-Lot0171501
Legal and Disclaimers
Legal and Disclaimers
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc
(NEB).
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain
additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at [email protected].
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.
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.