Exploring Wheat Germ Cell-Free Protein Expression

The article has been prepared and provided by CellFree Sciences.

Effective protein expression systems are essential for use of proteins in research and applied sciences. Apart from 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. The wheat germ cell-free protein expression system (WGCFS) developed at CellFree Sciences (CFS) allows to obtain high protein yields under reproducible conditions making it the method of choice for protein expression screens and large-scale protein production of up to hundreds of milligrams of protein per expression reaction.

As a classical expression system, wheat germ extracts had successfully been used over decades to express a wide range of proteins in many biochemical studies [1], arguing that the wheat germ system provides better folding properties for most proteins as compared to many other expression systems including proteins expressed in E.coli, or obtained from E. coli cell-free expression systems. Further improvements of the original wheat germ extracts were achieved in the laboratory of Prof. Endo at Ehime University in Japan leading to a highly optimized cell-free protein expression system for high-throughput protein expression at various scales [2]. Special washing steps were added during extract preparation to remove inhibitors of the translation reaction, RNases, and proteases. Those extracts allow for protein synthesis over an extended period of time without losing the RNA template or seeing degradation of the protein products.

Based on the methods of Ehime University, CFS developed commercial wheat germ extracts having largely increased protein yields as compared to the published protocols. Moreover, the original system was extended by developing dedicated wheat germ extracts for the preparation of His- or GST-tagged proteins. 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 [3]. Our WGCFS was used in many proteomic studies, because of its unmatched success rate for expressing multiple proteins on a large-scale. This high performance of our WGCFS has been demonstrated, for example, during its use at the so-called “Human Protein Factory” [4]. The project targeted at the expression of 13,364 human proteins, where 12,996 of their clones produced protein in the WGCFS (97.2%); out of those 12,682 were found in the soluble fraction when expressed with a GST-tag. These proteins are now the basis for preparing protein arrays covering nearly the entire human proteome. Similarly, the WGCFS from CFS has been used by commercial providers to prepare many thousands of antigens for antibody preparation. Even extremely difficult to express proteins, like for example malaria proteins for vaccine development, could be prepared by the WGCFS on a large-scale with a very good success rate [5].

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1: Premium PLUS Expression Kit and reaction vials with seal for bilayer reaction

With the Premium PLUS Expression Kit (Figure 1A), CFS wants to give customers the opportunity to establish our WGCFS for work at their laboratories. The kit contains expression vector pEU-E01-MCS for preparing an expression template, a positive control (DHFR inserted in vector pEU-E01-MCS), and ready-to-use premixed reagents to conduct 8 protein expression experiments. Reagents are provided in color-coded tubes, and the Translation Buffer is already added to the reaction cups for the bilayer reaction format discussed below (Figure 1B). Moreover, primers are provided to prepare expression templates by PCR avoiding complicated cloning steps during quick protein expression testing. In our WGCFS, proteins are prepared in a two-step process (Figure 2), 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.

 

 

 

 

 

 

 

 

 

 

Figure 2: Experimental flow and time requirements for protein expression reaction (reagents indicated in bold are provided with the Premium PLUS Expression Kit)

For working on a preparative scale, it proved to be beneficial to separate the transcription and translation reactions, because optimal reaction conditions can be used for both steps. In particularly, reducing the reaction temperature during protein synthesis has greatly improved the yields of soluble proteins (see data in Figure 4C below). Well expressed proteins can easily be detected by SDS page as distinct bands as compared to a negative control containing only the proteins from the wheat germ extract as shown in Figure 3 for the DHFR protein provided with the Premium PLUS Expression Kit as a positive control. Alternatively, protein expression can be tested by adding a tRNA with a fluorescently-labeled lysine to the translation reaction. The randomly incorporated fluorescent lysines will yield in labeled proteins that can easily be detected with a fluorescent scanner. Since only the newly synthesized protein will contain the label, no other proteins are detected. The fluorescent label is highly sensitive, and even very small amounts of proteins can be detected in this way.

 

 

 

 

 

 

 

 

 

 

Figure 3: SDS page confirming expression of the DHFR positive control protein

Standard cell-free protein translation reactions commonly reach their highest yields 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) [6]. A bilayer reaction is setup by placing the wheat germ extract together with the RNA under a top layer with the reaction buffer. 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, the bilayer format commonly provides higher protein yields than possible in a simple batch reaction format (Figure 4C). As shown in Figure 4A, bilayer reactions should be setup in the flat bottom vials provided with the Premium PLUS Expression Kit (Figure 1B). For incubation, those can easily being sealed with the sealing tape included in the kit. If flat bottom vials are unavailable, we recommend the use of different multi-well plate formats for reaction setup, where each well has a flat bottom. Multi-well plates are very convenient for performing many translation reactions in parallel as for examples needed during screening experiments.

 

 

 

 

 

 

 

 

 

 

 

Figure 4: Bilayer reaction format for cell-free protein translation reactions (refer to text for further details)

Bilayer reactions have been up scaled up to a 6 ml reaction volume used in the CFS Protemist® DTII fully automated protein synthesizer. This instrument can perform all steps for providing tag-purified proteins starting directly from a DNA template [3]. 

In contrast to cell-based expression systems, WGCFS allows for easy manipulation of the reaction conditions. Therefore different 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 proteins. Similarly, liposomes have been added to the expression reaction for direct preparation of proteoliposomes for working on membrane proteins [7]. Another example is the recently published expression of holomyoglobin in the presence of added hemin [8].

 

 

 

 

 

 

 

 

 

 

Figure 5: Dialysis reaction formats for working on different reaction scales

While the bilayer reaction format is optimal for quick reaction setup and effective small-scale reactions, protein yields can be further improved by continuous buffer exchange in a dialysis reaction format (Figure 5). Under standard conditions, we perform dialysis translation reactions for up to 72 hours at 15°. Table 1 provides normalized protein yields per ml of the CFS standard wheat germ extract WEPRO7240 and 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 (CFS standard protocol). Thus such a reaction can, for example, easily provide in most cases sufficient amounts of protein for rapid antigen preparation for immunization experiments and the development of immune assays.

 

 

 

Table 1: Normalized protein yields for different reaction cell-free protein translation reaction formats

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 within the Premium PLUS Expression Kit with our Protein Research Kits for easy setup of 24 bilayer reactions per kit. In addition, CFS offers Protein Expression Kits for manual use or use on a Protemist® DTII instrument to run bilayer reactions on different reaction scales. 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, where CFS provides user protocols for performing dialysis reactions instead of the bilayer method.

For working with our protein expression system on a routine basis, we recommend the following considerations for setting up production of individual proteins (Figure 6):

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 Figure 6: Process for setting up WGCFS protein expression pipeline

  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). Other affinity tags have been successfully applied to our WGCFS. Additional expression vectors for WGCFS are available in the public domain as for example at the depository of the PSI project (https://dnasu.org/DNASU/).
  2. The cDNA for the protein of interest may be obtained by PCR, cDNA cloning, or gene synthesis. The WGCFS does not require codon optimization, and therefore many cDNAs from public depositories have been successfully expressed in our expression system (e.g. RIKEN mouse FANTOM clones, RIKEN Arabidopsis clones, or clones from the Mammalian Gene Collection). However, many gene synthesis companies provide the option to optimize sequences for use in WGCFS, an option that could be used if the cDNA is not needed for any other purpose than working with the WGCFS. PCR products maybe directly used in the WGCFS, where CFS can provide a protocol and primer information for introducing the SP6 promoter and enhancer sequence in a two-step overlap extension PCR (so-called “Split PCR method”). Similarly, the short sequence for a His-tag can be introduced during PCR. We advise to confirm the sequence of the expression template prior to use in protein synthesis.
  3. We recommend testing the expression template, where high quality DNA preparations are needed for successful protein synthesis (e.g. prepared by use of a commercial plasmid DNA purification kit). Besides determining the OD of the DNA template, small expression reactions in the presence of a fluorescent lysine can be used to confirm that the protein is made from a given template. We advise to test each expression template before up scaling protein production.
  4. Select a reaction format depending on your protein needs. While the bilayer reaction format is our standard reaction, we advise for large-scale protein production to use a dialysis reaction. For frequent users like for example service laboratories or protein research centers, the WGCFS can be fully automated as for example in the Protemist® DTII instrument available from CFS.
  5. 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 spectroscopy experiments. For some of those needs CFS offers dedicated reagents, already including lyophilized liposomes or isotope-labeled amino acids.

Many thousands of proteins from microorganisms to mammalians have been successfully produced by our WGCFS, among others, in large-scale protein crystallography [9] and NMR studies [10], preparation of standards for mass spectroscopy [11], search for new enzymatic functions [12], antigen production [13], and protein-protein interaction screening [14]. 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 you an entry kit to explore the use WGCFS for the expression of your proteins at need.

Refer to our homepage at http://www.cfsciences.com/eg/ for more information on our products and services, or contact us directly at tech-sales@cfsciences.com for assistance.

 

Use our exclusive offer presented at flash4science to try Premium PLUS Expression Kit for free. Share it linkedIn, facebook, twitter, google+.

 

Selected references

  1. Harbers, M., Wheat germ systems for cell-free protein expression. FEBS letters, 2014. 588(17): p. 2762-73.
  2. 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.
  3. Beebe, E.T., et al., Automated cell-free protein production methods for structural studies. Methods in molecular biology, 2014. 1140: p. 117-35.
  4. 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.
  5. 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.
  6. 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.
  7. 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.
  8. 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.
  9. Watanabe, M., et al., Cell-free protein synthesis for structure determination by X-ray crystallography. Methods in molecular biology, 2010. 607: p. 149-60.
  10. Makino, S., et al., Cell-free protein synthesis for functional and structural studies. Methods in molecular biology, 2014. 1091: p. 161-78.
  11. Singh, S., et al., A practical guide to the FLEXIQuant method. Methods in molecular biology, 2012. 893: p. 295-319.
  12. Takasuka, T.E., et al., Cell-free translation of biofuel enzymes. Methods in molecular biology, 2014. 1118: p. 71-95.
  13. Matsunaga, S., et al., Wheat germ cell-free system-based production of hemagglutinin-neuraminidase glycoprotein of human parainfluenza virus type 3 for generation and characterization of monoclonal antibody. Frontiers in microbiology, 2014. 5: p. 208.
  14. Kasahara, K., et al., Ubiquitin-proteasome system controls ciliogenesis at the initial step of axoneme extension. Nature communications, 2014. 5: p. 5081.

 

 

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