Skip to content

Basket

You currently have no items in your basket.

CellFree Sciences

CellFree Sciences Co, established since 2002, started creating their products in the labs of Ehime University, Japan. Expanding their work through research, setting up products for various well known international Universities and Laboratories. Strategically partnering with leading providers of integrated software Biotechnology solutions, introduced in MARKET ProtemistR and WEPROR and many more.

CellFree Sciences

To view our range of products, click here

View Products

Why Choose CFS?

A beginners guide...

Further information

FLEXIQuant Protein Expression Kit

Wheat Germ Cell-Free Expression System to Make Full-Length Isotope-Labeled MS Standards

Click Here

Introduction to Wheat Germ Cell-Free Protein Expression

 

The wheat germ cell-free protein expression system (WGCFS) has been used for many years as a basic tool to drive life science research and to support product development in industry. Below, we are providing a general introduction on how to use WGCFS.

Overview

Effective protein expression systems are essential for using proteins in research and applied sciences. Apart from the common approaches used for cell-based protein synthesis, cell-free protein expression systems are important because of their ability to rapidly produce proteins directly from a DNA template [1,2]. The WGCFS developed at CellFree Sciences (CFS) allows further to obtain high protein yields under reproducible conditions making it one of the methods of choice for protein expression screens and large-scale protein production of up to hundreds of milligrams of protein per expression reaction.

The WGCFS from CFS

As a classical expression system, wheat germ extracts have successfully been used over decades to express a wide range of proteins in many biochemical studies, arguing that the wheat germ system provides better folding properties for most proteins than several other expression systems including proteins expressed in E.coli, or obtained from E. coli cell-free expression systems [3,4] . Further improvements of the original wheat germ extracts were achieved in the laboratory of Prof. Y. Endo at Ehime University in Japan leading to a highly optimized cell-free protein expression system for high-throughput protein expression at various scales [5]. Special washing steps were added during extract preparation to remove inhibitors of the translation reaction, RNases, and proteases [6]. Those extracts allow for protein synthesis over extended time without losing the RNA template or seeing degradation of the protein products.

Based on the published methods from Ehime University, CFS developed commercial wheat germ extracts (WEPRO®) offering largely increased protein yields as compared to the regular wheat germ extracts on the market. Moreover, the original system was extended by developing dedicated wheat germ extracts for the preparation of His- or GST-tagged proteins (H and G versions) allowing for higher protein purity after purification on a Ni or glutathione column respectively. In combination with other improvements, CFS now provides high-performance protein expression systems for manual and fully automated, cell-free production of native, as well as His- or GST-tagged proteins at high yield and purity. Our WGCFS is used in many proteomic studies, because of its unmatched success rate for high-throughput protein expression, protein co-expression studies, and upscaled protein production for large-scale needs. The high performance of our WGCFS has been demonstrated, for example, during its use at the so-called “Human Protein Factory”[7] . . In this project, expression templates for the WGCFS were prepared by PCR and then directly used for protein expression experiments further streamlining the process for analyzing many proteins.  The project targeted at the expression of 13,364 human proteins, where 12,996 of their clones produced a protein in the WGCFS (97.2%); out of those 12,682 were found in the soluble fraction when expressed together with a GST-tag. These proteins are now the basis for preparing protein arrays covering nearly the entire human proteome (“Protein Active Arrays”) that are used for the identification of biomarkers related to cancer and autoimmune diseases. Similarly, the WGCFS from CFS has been used in-house and by commercial providers to prepare thousands of antigens for antibody preparation, many of those antigens are now produced by on-demand services on customer request. Even extremely difficult to express proteins, like for example malaria proteins for vaccine development and biomarker discovery, could be prepared by our WGCFS on a large-scale at a very good success rate [8,9].

WGCFS Entry Kits

CFS is providing easy to use entry kits to serve needs in regular research applications.

Figure 1: CFS Premium PLUS Expression Kit (A) and ready-to-use reaction cups for bilayer reactions provided with the kit (B). Reagent vials are color coded as indicated on the label.

With the Premium PLUS Expression Kit, CFS wants to give customers an opportunity to establish our WGCFS for projects at their laboratory (Figure 1). The kit contains the general expression vector pEU-E01-MCS for preparing an expression template, a positive control (DHFR cDNA inserted in vector pEU-E01-MCS), and ready-to-use premixed reagents to conduct 8 protein expression experiments using a bilayer reaction format. Moreover, primers are provided to prepare expression templates by PCR avoiding complicated cloning steps for quick protein expression testing. Proteins are prepared in a two-step process, where in the first step the SP6 RNA polymerase is used to obtain RNA transcripts from the expression template. In the second step, these RNA transcripts are then utilized in the protein synthesis reaction. As shown in Figure 2, the pre-mixed reagents allow to setup each reaction by simple pipetting steps.

Figure 2: Experimental flow and time requirements for protein expression reaction.

For better protein yields, it proved beneficial to separate the transcription and translation reactions, because optimal reaction conditions can be used for each step. In particular reducing the reaction temperature during protein synthesis has greatly improved the yields of soluble proteins (see Figure 4C below). Well expressed proteins can easily be detected on SDS-PAGE as distinct bands as compared to a negative control containing only the proteins from the wheat germ extract as shown in Figure 3A for the DHFR protein used as a positive control in the Premium PLUS Expression Kit. Alternatively, protein expression can be tested by adding a tRNA with a fluorescently-labeled lysine (not available from CFS) to the translation reaction. The randomly incorporated fluorescent lysines will yield in labeled proteins that can easily be detected on an SDS-PAGE using a fluorescent scanner. Since only the newly synthesized protein will contain the label, no other proteins from the wheat germ extract are detected as shown in Figure 3B using the DHFR positive control as an example. The fluorescent label is highly sensitive, and even very small amounts of proteins can be detected in this way. Therefore, using such a label can be an immense help when testing an expression vector for the first time, or when handling many samples. Also, Western Blotting (e.g. using an antibody against a tag) can improve the sensitivity and specificity when testing protein expression from a new template.  CFS can provide a standard protocol for using the labeling method to perform for example quick quality tests on new expression vectors.

Figure 3: SDS-PAGE confirming expression of the DHFR positive control protein in a regular expression reaction (A) and detected by incorporation of fluorescent lysine (B). M: Molecular weight marker; 1: Negative control is expression reaction without template; 2: Positive control is expression reaction with template for the DHFR protein; orange arrows indicate DHFR protein band; red arrow indicates free label.

CFS Bilayer Reaction Format

Standard cell-free protein expression reactions in a batch format commonly reach their highest yield after a few hours because the substrates are exhausted and inhibitors, mostly phosphate, hamper further protein synthesis. Therefore, it is preferable to apply reaction formats, which slowly supply the translation reaction with substrates, or even exchange the entire reaction buffer over time to remove also inhibitors of the translation reaction. For small-scale reactions, this can be achieved by the so-called bilayer method (Figure 4) [10].

A bilayer reaction is setup by placing the wheat germ extract together with the RNA template from the transcription reaction under a top layer containing the high-performance reaction buffer SUB-AMIX SGC®. This can be easily achieved because of the much higher density of the wheat germ extract as compared to the reaction buffer (Figure 4A). After setting up the bilayer reaction, both layers mix slowly during incubation allowing for protein expression reactions of up to 24 hours as shown for the expression of a functional GFP protein in Figure 4B. While delaying the protein expression reaction in the beginning, the bilayer format commonly provides higher protein yields after up to 24 h reaction time than possible in a simple batch reaction format (Figure 4C). As shown in Figure 4A, bilayer reactions should be setup in flat bottom reaction vials as for example the ones already provided with the Premium PLUS Expression Kit (Figure 1B). The flat bottom reaction vials provided with the Premium PLUS Expression Kit already contain the high performance SUB-AMIX SGC® reaction buffer to further support setting up the bilayer reaction with a quick pipetting step. Otherwise, we recommend the use of multi-well plate formats for reaction setup, where the wells should have a flat bottom. Multi-well plates are very convenient for performing many translation reactions in parallel and can be obtained with different well sizes. As indicated above, the protein expression reaction is best performed around 15°C to achieve higher protein yields. We observed reduced protein yields after increasing the reaction temperature, where wheat derived ribosomes do not work well at 37°C, the temperature used for RNA transcription reactions (see Figure 4C showing GFP yields obtained at 16°C and 26°C).

Figure 4: Bilayer reaction format for cell-free protein translation reactions (refer to text for further details on panels A to C). Image in B provided by Prof. Y. Endo.

Bilayer reactions have been upscaled up to a 6 ml reaction volume used in the CFS Protemist® DTII fully automated protein synthesizer [11].

This instrument can perform all steps for providing tag-purified proteins starting directly from a DNA template. The Protemist® DTII instrument is for example used to prepare proteins for structural genomics projects, in screening experiments to find new enzymatic activities [12], or on-demand protein expression services offered by CFS.

Using Open Reaction Format

In contrast to cell-based expression systems, WGCFS allows for easy manipulation of the reaction conditions. Therefore, various expression conditions can be tested in parallel without need for preparing different expression templates or cell systems. This is very useful when working for example with detergents to increase protein solubility. In our experience, especially detergent Brij-35 (a polyoxyethylene alkyl-ether) at concentrations of 0.025 to 0.05 % w/v has been well tolerated in the translation reactions while often improving the yields of soluble protein. Using the open nature of the cell-free protein expression system, CFS offers a special reagent kit for the incorporation of 13C/15N isotope-labeled lysine and arginine to obtain protein standards for MRM/SRM mass spectrometry (Premium PLUS Expression Kit for MS), and to support FLEXIQuant experiments [13, 15] using a peptide related to a tag for protein quantification (FLEXIQuant PLUS Expression Kit). Similarly, liposomes have been added to the expression reactions for direct preparation of proteoliposomes containing membrane proteins. In particular asolectin from soybeans has been successfully used in this context. CFS offers a special starter kit for the preparation of proteoliposomes to assist studies on membrane proteins (ProteoLiposome PLUS Expression Kit) along with the ProteoLiposome BD Expression Kit to further upscale the production of membrane proteins. The ProteoLiposome PLUS Expression Kit uses a small-scale bilayer reaction and provides a wheat germ extract that contains already asolectin-derived liposomes. Other examples for the flexibility of the expression system and the use of cofactors have been described in the literature, such as the expression of holomyoglobin in the presence of added hemin [16], or the use of chaperons to support disulfide bond formation [17]. 

Using Different Reaction Formats

While the bilayer reaction format is optimal for quick reaction setup and effective small-scale expression reactions, protein yields can be further improved by continuous buffer exchange in a dialysis reaction format.

Reaction methods

Batch

Bilayer

Dialysis

Yield per ml WEPRO®

~1.6 mg

~3.2 mg

~20 mg

 

Figure 5: Normalized protein yields for different cell-free protein translation reaction formats.

Dialysis reactions are preferable when larger protein amounts are needed. Under standard conditions, we perform dialysis translation reactions for up to 72 hours at 15°C. Figure 5 provides normalized protein yields per one ml of the CFS standard wheat germ extract WEPRO®7240 using different reaction formats for the expression of a functional GFP protein. The data show the clear advantage of the dialysis method for preparative protein expression experiments, where about 9 mg of crude GFP protein can be obtained from a 3 ml dialysis reaction. Thus, such a reaction can for example easily provide in most cases sufficient amounts of protein for rapid antigen preparation in immunization experiments and the development of immune assays. Also our ProteoLiposome BD Expression Kit mentioned above uses a modified dialysis protocol and lyophilized liposomes for large-scale preparation of proteoliposomes, e.g. to prepare antigens for antibody generation [18]. 

Figure 6: Setup of protein expression reactions using a dialysis format: 50 µl reaction scale using a dialysis cup (A); 3 ml reaction scale using a dialysis slider cassette (B).

CFS provides standard user protocols for performing dialysis reactions on a 50 µl or 3 ml reaction scale (Figure 6) using dialysis cups or dialysis slider cassettes depending on the reaction size. For even larger protein needs, CFS is offering the Protemist® XE instrument for automated refeeding assays that in principle allow to produce up to 1 g of crude protein per run.

CFS Protein Expression Kits

The Premium PLUS Expression Kit provides an easy format to test proteins for expression in our system using bilayer reactions. We provide the same ready-to-use reagents from the Premium PLUS Expression Kit with our Protein Research Kits for easy setup of 24 bilayer reactions per kit. Protein Research Kits are available containing the standard wheat germ extract (S version), and in H and G versions for working with His- or GST-tagged proteins. We are also using the same kit format in our labeling kits for protein MS and the small-scale preparation of proteoliposomes. In addition to the premixed reagent kits, CFS offers large-scale Protein Expression Kits for manual use or for use on a Protemist® DTII protein expression instrument to run bilayer reactions on different reaction scales. Protein Expression Kits are available for all versions of our wheat germ extracts including amino acid free reagents for the preparation of labeled protein, e.g. for use in protein NMR. As part of our batch program, all our protein expression reagents can also be purchased individually to give customers full flexibility to plan their experiments on the scale at need for their studies. In this way, the customer can decide on the use of the most suitable reaction format and wheat germ extract.

Setting Up Protein Expression Experiments

CFS has been using the WGCFS over many years to prepare thousands of proteins inhouse, mostly for use as antigens in antibody preparation. Based on our routines, we recommend the following considerations for setting up the production of individual proteins and for working with our protein expression system on a routine basis (Figure 7).

  1. Plan your experiments ahead and decide in advance on how you would like to obtain your protein, e.g. in its native form or as a fusion protein using an affinity tag. Refer to general advice in the literature on designing expression templates for protein expression based on sequence information in public databases and available tools for protein and sequence analysis. Confirm before preparing the actual expression template that your template is correct and has a starting ATG and stop codon in line with the open reading frame you want to use. If the template will be made by gene synthesis, consider the option to optimize the sequence for expression in wheat; it may help to get better protein yields. Gene synthesis providers commonly offer this option.

Figure 7: Workflow to set up a cell-free protein expression experiment.

  1. We recommend using dedicated expression vectors for protein expression. While templates can be made directly by PCR, working with an expression vector will provide higher yields and more reproducible results. CFS provides dedicated expression vectors for our WGCFS for the preparation of native proteins or working with different affinity tags (e.g. His- or GST-tag). In additions, several expression vectors for WGCFS are available in the public domain as for example at the depository of the PSI project (https://dnasu.org/DNASU/). However, CFS can provide protocols to prepare templates by PCR in case this is more suitable for your project.
  2. We recommend testing the expression template, where high quality DNA preparations are needed for successful protein synthesis (e.g. obtained by use of a commercial DNA purification kit). Besides determining the OD of the DNA template, small expression reactions, e.g. in the presence of a fluorescent lysine, can be used to confirm that the protein can be made from a given template. We advise to test each expression template before upscaling protein production. It is better to use a highly sensitive detection method like the fluorescent lysine method at this point as it will make signal detection and analysis easier. You may also be able to work on a smaller reaction scale than needed to detect a protein by staining a regular SDS-PAGE.
  3. Select a reaction format depending on your protein needs. While the bilayer reaction format is our standard reaction for small scale and high-throughput applications, we advise for large-scale protein production to use a dialysis reaction. It is better to keep the reaction format from testing to scaling as within the same format the yields are more predictable. For frequent users like for example service laboratories or protein research centers, the WGCFS can be fully automated as for example done in the Protemist® DTII instrument available from CFS.
  4. Additional considerations may apply for working with insoluble proteins that could benefit from the use of detergents. However, many more options are available when using WGCFS as for example when working with proteoliposomes or preparing isotope-labeled proteins for NMR and mass spectrometry experiments. For some of those CFS offers dedicated reagents, e.g. amino acid free buffers and wheat germ extracts (WEPRO®8240 series), or kits already including lyophilized liposomes or isotope-labeled amino acids. Refer to the literature for more examples on different cofactors that have been used in cell-free protein expression systems.

 

Many thousands of proteins from microorganisms to mammalians have been successfully produced by our WGCFS, among others, in large protein crystallography and NMR studies [19] , preparation of standards for mass spectroscopy [14, 15. 20, 21], search for enzymatic functions [12], protein engineering, antigen production, analysis and use of viral proteins [22] , screening compound libraries for drug targets and agrochemicals [23], and protein-protein interaction screening [24]. Therefore, we are confident that our WGCFS can be applied for a wide range of protein studies in basic research and applied sciences. With the Premium PLUS Expression Kit, CFS is offering an entry kit to explore the use WGCFS for the expression of your proteins at need. Explore this opportunity to try wheat germ cell-free protein expression or contact us on other opportunities to establish our expression system at your facilities.

 

Selected References

1.             Carlson, E.D., et al., Cell-free protein synthesis: applications come of age. Biotechnology advances, 2012. 30(5): p. 1185-94.

2.             Zemella, A., et al., Cell-Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems. Chembiochem, 2015. 16(17): p. 2420-31.

3.             Harbers, M., Wheat germ systems for cell-free protein expression. FEBS letters, 2014. 588(17): p. 2762-73.

4.             Sawasaki, T., et al., A cell-free protein synthesis system for high-throughput proteomics. Proceedings of the National Academy of Sciences of the United States of America, 2002. 99(23): p. 14652-7.

5.             Madin, K., et al., A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: plants apparently contain a suicide system directed at ribosomes. Proceedings of the National Academy of Sciences of the United States of America, 2000. 97(2): p. 559-64.

6.             Takai, K., T. Sawasaki, and Y. Endo, Practical cell-free protein synthesis system using purified wheat embryos. Nature protocols, 2010. 5(2): p. 227-38.

7.             Goshima, N., et al., Human protein factory for converting the transcriptome into an in vitro-expressed proteome. Nature methods, 2008. 5(12): p. 1011-7.

8.             Arumugam, T.U., et al., Application of wheat germ cell-free protein expression system for novel malaria vaccine candidate discovery. Expert review of vaccines, 2014. 13(1): p. 75-85.

9.             Kanoi, B.N., et al., Antibody profiles to wheat germ cell-free system synthesized Plasmodium falciparum proteins correlate with protection from symptomatic malaria in Uganda. Vaccine, 2017. 35(6): p. 873-881.

10.           Sawasaki, T., et al., A bilayer cell-free protein synthesis system for high-throughput screening of gene products. FEBS letters, 2002. 514(1): p. 102-5.

11.           Beebe, E.T., et al., Automated cell-free protein production methods for structural studies. Methods in molecular biology, 2014. 1140: p. 117-35.

12.           Takasuka, T.E., et al., Cell-free translation of biofuel enzymes. Methods in molecular biology, 2014. 1118: p. 71-95.

13.           Singh, S., et al., A practical guide to the FLEXIQuant method. Methods in molecular biology, 2012. 893: p. 295-319.

14.           Singh, S., et al., FLEXIQuant: a novel tool for the absolute quantification of proteins, and the simultaneous identification and quantification of potentially modified peptides. Journal of proteome research, 2009. 8(5): p. 2201-10.

15.           Singh, S.A., et al., FLEXIQinase, a mass spectrometry-based assay, to unveil multikinase mechanisms. Nature methods, 2012. 9(5): p. 504-8.

16.           Samuel, P.P., et al., Apoglobin Stability Is the Major Factor Governing both Cell-Free and In Vivo Expression of Holomyoglobin. The Journal of biological chemistry, 2015.

17.           Gad, W., et al., The quiescin sulfhydryl oxidase (hQSOX1b) tunes the expression of resistin-like molecule alpha (RELM-alpha or mFIZZ1) in a wheat germ cell-free extract. PloS one, 2013. 8(1): p. e55621.

18.           Takeda, H., et al., Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay. Scientific reports, 2015. 5: p. 11333.

19.           Makino, S., et al., Cell-free protein synthesis for functional and structural studies. Methods in molecular biology, 2014. 1091: p. 161-78.

20.           Takemori, N., et al., High-throughput synthesis of stable isotope-labeled transmembrane proteins for targeted transmembrane proteomics using a wheat germ cell-free protein synthesis system. Molecular bioSystems, 2015. 11(2): p. 361-5.

21.           Takemori, N., et al., High-throughput production of a stable isotope-labeled peptide library for targeted proteomics using a wheat germ cell-free synthesis system. Molecular bioSystems, 2016.

22.           Takahashi, H., et al., Establishment of a robust dengue virus NS3-NS5 binding assay for identification of protein-protein interaction inhibitors. Antiviral research, 2012. 96(3): p. 305-14.

23.           Nemoto, K., et al., Identification of new abscisic acid receptor agonists using a wheat cell-free based drug screening system. Sci Rep, 2018. 8(1): p. 4268.

24.           Kasahara, K., et al., Ubiquitin-proteasome system controls ciliogenesis at the initial step of axoneme extension. Nature communications, 2014. 5: p. 5081.

This website uses cookies. For more information about these please click here.
By continuing to browse you consent to the use of cookies