- What does ENDEXT Technology signify?
ENDEXT® Technology signifies the entirety of the technology that supports the wheat germ
cell-free translational system developed by Professor Yaeta Endo of Ehime University. Professor Endo and his co-workers discovered that translation in wheat germ extract was inhibited by endogenous inhibitors such as RIP (ribosome inactivating proteins), thionine, and proteases, and that their removal from wheat embryos resulted in a robust and highly efficient cell-free expression system. Following this discovery, they developed a high-throughput protein synthesis system to produce not only authentic proteins but also proteins labeled with biotin, fluorescent substrates, stable isotopes, or selenomethionine. Derived from the eukaryotic cell, the wheat germ cell-free system is capable of producing in proper folding proteins ranging from those of microorganisms to multidomain proteins found in higher organisms.
- What are the advantages of the wheat germ expression system compared with other systems for synthesizing recombinant proteins?
High quality protein synthesis in a wide temperature range:
Compared with those produced in conventional cell lysates, the proteins synthesized in the wheat germ cell-free expression system are more likely to be properly folded and soluble. Furthermore, they are much less prone to degrade, because the system contains only negligible amounts of endogenous proteases. In addition, the system permits translation in a wide temperature range. The optimum translation temperature lies between 15 ºC and 26 ºC. In this temperature range, protein yield is nearly constant. Translation can also be carried out at temperatures lower than 15 ºC. This makes it possible to synthesize proteases, which are self-destructive at room temperature, and proteins that lack high-temperature stability.
No necessity for codon optimization:
The system shows little codon usage preference. As a result, even proteins encoded by AT-rich cDNAs, e.g., malaria proteins, can be synthesized without codon optimization.
Fewer process steps:
The system requires fewer process steps. It requires no lysis steps such as sample sonication, detergent lysis, lysozyme and/or nuclease treatment. Centrifugation of crude lysate is not necessary, because less insolubles are contained in the wheat germ extract than in cell extracts. In fact, our automatic synthesizer Protemist DT II performs His or GST tag affinity purification without centrifugation.
Ease of use & cost-effectiveness:
The system requires minimal technical training and skill required for successful protein expression. For many proteins, the system is more cost-effective than insect or mammalian cell expression systems, as it requires no tissue culture facilities.
High reproducibility:
Protein expression in the system is highly reproducible. This contrasts with protein expression in in vivo systems, whose reproducibility is affected by a variety of factors such as media quality, contamination, and toxicity of target proteins.
Lack of glycosylation (characteristic):
No post-translational glycosylation takes place in the system. This can be exploited for the production of unglycosylated proteins such as malaria proteins and homogenous protein populations as desirable for X-ray crystallography.
- What kinds of proteins have been synthesized in the wheat germ cell-free expression system?
CFS and Ehime University have collectively synthesized more than 6,000 proteins, which include nuclear receptors (e.g., ER), transcriptional factors (e.g., NF-κB), Ser/Thr kinases (p38α, CaMK, etc.), Tyr kinases (JAK, TIE2, etc.), proteases (caspases, SARS protease, etc.), pathogenic proteins (e.g., malaria plasmodium proteins), and others. Not subjected to species limitation, the wheat germ cell-free expression system has been used to synthesize a broad range of proteins, from proteins of microorganisms (bacteria, thermophiles, pathogens, etc.) to those of higher forms of life (plants, fish, and mammals). Certain membrane proteins have been synthesized in a soluble form, if yields are low.
- What are the size limits of proteins synthesized in the system?
Proteins ranging from 10 kDa to 360 kDa have been successfully expressed in the system. Proteins (peptides) of lower molecular weights have also been expressed by inserting tandem DNA repeats in the MCS of pEU vector or fusing GST tag.
- What is the typical protein yield in the system?
The yield ranges from nanogram to milligram per reaction, depending on proteins, translation reaction scale, and translation reaction methods. ENDEXT® Technology employs two methods for translation, the repeat batch and bi-layer methods. In the bi-layer method, the reaction scale ranges from 150 µl to 6 ml, the maximum yield in a 6-ml reaction about 0.5 mg.
- Which reagents does CFS supply, and which reagents should the user prepare?
CFS supplies expression kits and individual reagents for transcription and translation. Our kits contain pEU plasmid, pEU/DHFR control, wheat germ extract (WEPROs), translation buffer (SUB-AMIX), and transcription reagents. All the reagents can be purchased individually. The user is asked to prepare nuclease free water and a high-purity pEU plasmid template (OD260/OD280 = 1.70-1.85) containing a gene of interest or a PCR product ("split primer PCR", see Question 12). For more details about our kits and reagents, please visit our website.
- What experiences do CFS and its customers have in protein production and other applications?
Protein production:
• Large scale protein production for structural analysis by NMR or X-ray diffraction
• Large scale protein production of quality antigens for antibody preparation
• High-throughput protein production for functional analysis and screening
• Production of immunogenic malaria antigens that are difficult to express
• Recombinant single chain antibody production
Applications:
• Screening of cancer-related protein kinase substrates
• High-throughput analyses of interaction between nuclear receptors and target nucleotides
• Functional protein chips
• Protein modification for high performance
- Are there any requirements of the plasmid DNA to use as the template?
The plasmid vector pEU (EU for Ehime University) provided by CFS is specifically designed for the wheat germ cell-free expression system. The 5' and 3' sequences of pEU vector enhance mRNA translation. They permit the elimination of the 5' cap structure and poly(A) tail, which interfere with in vitro translation unless the capped mRNA concentration is controlled in a narrow range so that fragments of the capped mRNA may not inhibit translation initiation. With the problematic 5' cap structure and poly(A) tail eliminated, the wheat germ cell-free system does not require tedious optimization of mRNA concentration in translation.
High purity plasmid DNA is the key to successful transcription and subsequent translation in the wheat germ cell-free system. We therefore recommend that plasmids be purified with in vitro transcription grade kits of a reputable supplier and protected against RNase contamination. In general, plasmids that exhibit A260/A280 absorption ratios between 1.70 to 1.85 are considered to be of high quality.
- Where can the map and sequence of the pEU vector be found?
The map and sequence of the pEU vector can be found at:
http://www.cfsciences.com/eg/tech05.html
Vector Map
Sequence
- Can a different promoter other than SP6 be used for the wheat germ cell-free expression system?
In principle, we recommend the use of SP6 promoter inserted in the pEU vector. T7 promoter can be used, but the yield for the same mole of transcript would be lower. This is attributed to the difference in the 5' transcript site sequence between SP6 and T7 promoters.
- Can we use a PCR product as transcription template instead of pEU with a gene insert?
Yes, you can, using a method called "split primer PCR." In this method, you can make a linear DNA template for transcription by using our Wheat Germ Premium Expression Kit-PCR along with two custom primers procured by the user.
The split primer PCR consists of the following two steps:
1st PCR reaction: Use a custom sense primer containing a sequence derived from the cDNA of interest and a custom antisense primer derived from the plasmid containing the cDNA located about 1.6 knts downstream.
2nd PCR reaction: Use three primers, two sense and one antisense primers. The two sense primers are generic, made by adding the E01 enhancer and SP6 promoter to the 5' end of the PCR product. The antisense primer is the same as that used in the 1st PCR reaction. Since the SP6 promoter sequence is split between the two generic primers and is not entirely contained in either one, expression of non-specific amplification products is eliminated and production of the target protein is maximized.
Upstream of the ORF, the split primer PCR product contains the SP6 promoter and E01 translational enhancer sequences, and downstream of the ORF, more than 1.6 knts derived from the plasmid vector that contains the cDNA of interest. If a PCR product is not obtained from inserts in pET-24 and pET-28 series vector templates, an alternate vector context may be required (e.g., pUC, pBluesscript).
- Can we use His or GST tags on target proteins for purification?
Yes you can. Besides the standard wheat germ extract (WEPRO1240), we have two special types of wheat germ extracts made for obtaining higher degrees of purity from GST- and His-tagged proteins, respectively (WEPRO1240G for GST tag and WEPRO1240H for His tag). For the details of recommended manual purification protocols, please visit our website.
- Is it possible to use tags other than His or GST?
Although our experiences are mostly with synthesis and purification using GST- and His-tagged proteins, other tags such as FLAG, streptavidin, or lumio can also be used for the system.
- How should WEPRO be stored?
It should be shored at -80ºC. It should not be subjected to 3 or more freeze-thaw cycles. After the third freeze-thaw cycle, WEPRO’s protein synthesis potency may decrease, the degree of which depends on handling.
- What kinds of protein synthesis robots are available from CFS?
CFS markets three models for distinct purposes. They are all fully automatic robots:
• Protemist DT II is a versatile desktop robot, which performs transcription, translation, and purification. It can be operated either in a 6-well mode (6 ml/well) or 24-well (1.2 ml/well) mode.
• Protemist XE is a compact synthesizer for the large scale production of a single protein. It is under development as of Jan. 2009.
- What are the prices of your reagents, kits, and robotic synthesizers and how do we order?
Please contact our sales department at: tech-sales@cfsciences.com
- Do you offer protein synthesis as a service?
Yes we do. The scope of service is decided by selecting all or some of the tasks including plasmid construction, protein yield and solubility tests on a small scale, cost estimation,
and large-scale production with or without partial purification. Please feel free to ask to our sales department (tech-sales@cfsciences.com) for details.
- What are the primary references for this technology?
Basics:
- (1) Endo, Y. and Sawasaki, T. (2006) Cell-free expression systems for eukaryotic protein production. Curr. Opin. Biotechnol. 17 (4), 373-380.
- (2) Sawasaki, T., Ogasawara, T., Morishita, R. and Endo, Y. (2002) A cell-free protein synthesis system for high-throughput proteomics. Proc. Natl. Acad. Sci. USA 99, 14652-14657.
- (3) Madin, K., Sawasaki, T., Ogasawara, T. and Endo, Y. (2000) A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos. Proc. Natl. Acad. Sci. USA 97, 559-564.
Proteomics:
- (4) Endo, Y., and Sawasaki, T. (2004) High-throughput, genome-scale protein production method based on the wheat germ cell-free expression system. J. Struct. Funct. Genomics, 5, 45-57.
- (5) Sawasaki, T., Hasegawa, Y., Morishita, R., Seki, M., Shinozaki. K., and Endo, Y. (2004) Genome-scale, biochemical annotation method based on the wheat germ cell-free protein synthesis system. Phytochemistry, 65, 1549-1555.
NMR structural analysis:
- (6) Vinarov, D. A., Loushin Newman, C. L., and Markley, J. L. (2006) Wheat germ cell-free platform for eukaryotic protein production. FEBS J., 273, 4160-4169.
- (7) Vinarov D A, Lytle B L, Peterson F C, Tyler E M, Volkman B F, Markley J L. (2004) Cell-free protein production and labeling protocol for NMR-based structural proteomics. Nat Methods. Nov. 1 (2), 149-153.
X-ray crystallography:
- (8) Miyazono, K., Watanabe, M., Kosinski, J., Ishikawa, K., Kamo, M., Sawasaki, T., Nagata, K., Bujnicki, J.M., Endo, Y., Tanokura, M., and Kobayashi, I. (2007) Novel protein fold discovered in the PabI family of restriction enzymes. Nucleic Acids Res., 35, 1908-1918.
Synthesis and solubilizaion of membrane proteins:
- (9) Nozawa A, Nanamiya H, Miyata T, Linka N, Endo Y, Weber AP, Tozawa Y. (2007) A cell-free translation and proteoliposome reconstitution system for functional analysis of plant solute transporters. Plant Cell Physiol., 48, 1815-1820.
Disulfide bond formation:
- (10) Kawasaki, T., Gouda, M.D., Sawasaki, T., Takai, K., and Endo, Y. (2003) Efficient synthesis of a disulfide-containing protein through a batch cell-free system from wheat germ. Eur. J. Biochem., 270, 4780-4786.
Malaria proteins:
- (11) Takafumi Tsuboi, Satoru Takeo, Hideyuki Iriko, Ling Jin, Masateru Tsuchimochi, Shusaku Matsuda, Eun-Taek Han, Hitoshi Otsuki, Osamu Kaneko, Jetsumon Sattabongkot, Rachanee Udomsangpetch, Tatsuya Sawasaki, Motomi Torii, and Yaeta Endo (2008) The Wheat Germ Cell-Free Based Production of Malaria Proteins for Discovery of Novel Vaccine Candidates. Infect. Immun., 76 (4), 1702-1708.
Monograph:
- (12) Alexander S. Spirin and James R. Swartz (2007) Cell-free Protein Synthesis: Methods and Protocols. Vch Verlagsgesellschaft Mb
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